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UBC Theses and Dissertations

Isolation and characterization of plasmids carrying genes of bacteriophage T7 Smith, Richard D. 1978

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ISOLATION AND CHARACTERIZATION OF PLASMIDS CARRYING GENES OF BACTERIOPHAGE T7 by RICHARD D. SMITH B.Sc. (Honors, Biological Sciences) University of Cal i fornia - Irvine 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE FACULTY OF GRADUATE STUDIES in THE DEPARTMENT OF MICROBIOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November, 1978 (c) Richard D. Smith, 1978 In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i l ab le for reference and study. I further agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of this thes is for f inanc ia l gain sha l l not be allowed without my written permission. Department of The Univers i ty of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date c i - W , 1ST l < l > ? ABSTRACT The restr ict ion endonuclease Hpa I cleaves wild type T7 DNA into nineteen fragments. These Hpa I fragments were inserted into the ampici l l in resistance gene of the plasmid pBR322. The poly A - poly T method of construction was chosen in order to insure single T7 DNA inserts. Two hundred f i f ty clones were identified as carrying T7 DNA segments 32 + by colony hybridization with P T7 DNA. From these, clones carrying specific areas of the T7 genome were identified by various methods described in this thesis. Clones carrying Hpa I fragments E and G were identified by hybridization with Southern f i l t e r s of electrophoressed Hpa I digested T7 DNA. Clones carrying most of gene 5 and parts of gene 4 were identified by hybridization with a purified res tr ic t ion fragment carrying those genes (Hpa I fragment D). Clones containing parts of genes 10 and 11 were identified by marker rescue tests. Marker rescue "spot tests" were developed as a fast screening procedure for T7 genes and rely on generalized recombination between the T7 segment carried by the plasmid and the infecting phage DNA. i i i TABLE OF CONTENTS Page Abstract i i L i s t of Tables v Lis t of Figures v i L i s t of Abbreviations v i i Acknowledgements v i i i Introduction 1 Materials and Methods I. Bacterial Strains and Phage Stocks 6 II . Materials 7 III . Enzymes 7 IV. Restriction Endonuclease Digest Conditions 8 V. Preparation of Phage 8 VI. Isolation and Purif ication of Plasmid DNA's 9 VII. Extension of Linear pBR322 and T7 Hpa I Fragments 9 VIII. Annealing of Extended pBR322 and T7 DNA's 10 IX. Transformation with Annealed pBR322/T7 DNA 10 X. Preparation of Nitrocellulose Membranes 11 XI. Nick Translation of DNA's 11 32 XII. Hybridization of P-T7 DNA to Hognes or Southern F i l t ers 11 XIII. Marker Rescue Tests 12 XIV. Preparation of mRNA Fi l t ers and Hybridization 13 XV. Electroelution of DNA from Agarose Gels 13 XVI. Molecular Weight Analysis . . • 14 XVII. Containment 14 X V Page Results I. Principles of the Procedure 15 II . Digestion of pBR322 15 III . Digestion of T 7 + and VLG3 DNA 17 IV. Extension of DNA's with Homopolymer Blocks 20 V. Annealing 22 VI. Transformation 23 VII. Identification of a Clone Containing Hpa I Fragment E 26 VIII. The Cloning of Part ia l Hpa I D Fragments 26 IX. Cloning the Origin of Replication - Hpa I Fragment G 34 X. Additional Clones Carrying T7 Genes 36 Discussion 37 Bibliography 40 LIST OF FIGURES Figure T i t l e Page 1 Map of Bacteriophage T7 2 2 Map of pBR322 5 3 Outline of Cloning Procedure 16 4 Analysis of pBR322 on a 0.5% Agarose Gel 18 5 Hpa I Restriction Analysis of T7 and VLG3 19 6 Colony Hybridization 25 7 Hybridization of radioactive DNA's to NC F i l t er s . . . . 27 8 Recombination Spot Test of pRS 142 29 9 Colony Hybridization with Purified Hpa I Fragment D . . 31 10 Molecular Weight Analysis of Plasmids 33 11 Hybridization of mRNA F i l t er s 35 LIST OF TABLES Figure T i t l e Page 1 Molecular Weights of Hpa I Fragments and Cloned T7 Inserts 3 2 Transformation Frequencies with Varying Poly (A) - (T) Connector Lengths 24 3 Rescue Frequency of Amber Mutants by pRS142 and pBR322/C „ 28 Abbreviations used in this thesis are: LB : L-Broth Tc : Tetracycline Ap : Ampicillan NC : Nitrocellulose R.E. : Restriction Endonuclease CM : Chloramphenicol BSA : Bovine Serum Albumin (3ME : 2-mercaptoethanol DTT : Dithiothrei to l MOI : Mul t ip l i c i ty of Infection SSC : 0.15 M NaCl, 0.015 M Sodium Citrate ACKNOWLEDGEMENT S I wish to thank Dr. R. C. M i l l e r , J r . for his support and encouragement throughout this project. Many thanks go to Helen Smith and Deb Taylor for their assistance and helpful discussions. I am indebted to Michael Smith, Caroline Aste l l and Shirley Gillam for their advice and for supplying crucial enzymes used in this work. My thanks go to Dr. Ted Young and Susan Strome for teaching me valuable techniques used in the characterization of several plasmids. 1 INTRODUCTION Bacteriophage T7 was or ig inal ly characterized by Delbriick (7). The small size of i t s genome makes T7 an ideal subject for investigation into the biochemical processes that control the expression of i t s genetic material. The T7 genome was the f i r s t l inear , double stranded DNA to be transcribed in v i tro (6), provided the f i r s t example of post-transcript-ional modification of messenger RNA (9), and was the f i r s t phage to be packaged in v i tro (16). T7 DNA has been used for in v i tro recombination studies ( 2 4 ) . The genetic map of T7 has been nearly saturated with amber mutations. Two recent reviews concerning T7 were written by Studier (30, 32) . The T7 phage contains a l inear , double stranded DNA molecule with a molecular weight of 2.6 x 10^ daltons. There are 20 essential genes characterized by conditional lethal mutations. Five non-essential genes have been identified (31). Three classes of proteins have been determined by gel electrophoresis (30). Class I proteins, synthesized between 4 and 8 minutes post-infection are determined by genes 0.3 thru 1.3. Class II proteins are made between 6 and 15 minutes and are involved in DNA metabolism. This class includes the gene products from genes 1.7 to 6. Class III includes a l l remaining proteins; these are synthesized between 7 minutes and the time of l y s i s . Studier has determined the order of the Hpa I res tr ic t ion endo-nuclease fragments of T7 DNA (19). There are 19 fragments which range in molecular weight from 3.9 x 10 6 d to 0.19 x 10 6 d (Fig. 1, Table 1). The objective of this thesis was to insert specific Hpa I fragments into plasmids for use in future experiments concerning replication and 12 ,13 . 14 , 15 19 CO °" ^  0 0 :s CO I CO 0,3^ 0.7 .1 r.i i l l \ O C O CO rv_ CO co x ^ >> r o r O O CO CO LG3 •HH- •Tpf G Q C D O A RS L M N L , n r— r 1 1 1 i 1 1 O IO ? 0 30 40 50 6 0 70 80 90 IOC F i g u r e 1. Map of B a c t e r i o p h a g e T7 showing the p o s i t i o n o f the Hpa I r e s t r i c t i o n f r a g m e n t s ( m i d d l e ) i n r e l a t i o n t o the T7 genes ( t o p ) and the map p o s i t i o n ( b o t t o m ) . The a r r o w s i n d i c a t e p o s i t i o n . o f amber m u t a t i o n s ( 2 9 ) . 3 TABLE I THE MOLECULAR WEIGHTS OF HPA I FRAGMENTS AND T7 CLONED INSERTS — 6 Fragment . T7 units Base pairs M x 10 A 14.89 5956 3.94 B 10.92 4368 2.89 C 10.18 4072 2.70 D 9.55 3820 2.53 E 6.60 2640 1.75 F 6.25 2500 1.66 G 6.16 2464 1.63 H 5.78 2312 1.53 I 5.36 2144 1.42 J 5.29 2116 1.40 K 4.39 1756 1.16 L 3.46 1384 0.92 M • 2.49 996 0.66 N 2.23 892 0.59 0 2.10 840 0.56 P 1.51 604 0.40 Q 1.10 440 0.29 R 1.03 412 0.27 S 0.71 284 0.19 :als 100.00 40,000 26.49 Molecular lengths are given in T7 units (where 1 unit equals 1% of the length of T7 DNA), base-pairs, and as molecular weight (M ) of the sodium salt of DNA. The DNA of the T7 strain we use is taken to be 45,000 base-pairs long (Richardson, 1966), and the average molecular weight of a base-pair (sodium salt) is taken to be 662. (From Studier, ref. 19) CLONED FRAGMENTS Clone No. M x 10 pRS 136 IT 1.5 pRS 142 1.65 pRS 148 1.30 pRS 202 1.25 pRS 3 1.3 pRS 29 1.2 pRS 93 1.2 pRS - 88 0.7 6 Insert molecular weights were determined by linear regression analysis of agarose gels on a HP 9810 calculator. 4 recombination i n T7 phage. The vector chosen for i n s e r t i n g these fragments, pBR322, contains genes for A m p i c i l l i n and T e t r a c y c l i n e resistance. Various si n g l e r e s t r i c t i o n s i t e s within these drug resistance genes make pBR322 an i d e a l cloning v e h i c l e (Fig. 2). The poly A - poly T connector method was chosen for T7 fragment i n s e r t i o n . The method u t i l i z e s homopolymer segments of poly A on the plasmid molecule and segments of poly T on the T7 DNA to be inserted. Upon annealing, recombinant molecules are formed. By t h i s method of construction, a l l clones carrying T7 DNA have sin g l e i n s e r t s . 6 MATERIALS AND METHODS B a c t e r i a l S t r a i n s and Phage Stocks The E_. c o l i s t r a i n s B23 (su°) and 0-11 1 (su-j--r) were used as non-permissive and permissive T7 h o s t s , r e s p e c t i v e l y (29) . E_. c o l i s t r a i n BL-2 was used as the non-permissive host f o r amber mutants i n T7 gene 1.3 (31). BR3-4 was i s o l a t e d by K. Burck from the Studier s t r a i n BR-3 and proved to be a b e t t e r non-permissive host f o r amber mutations i n genes 0.3 and 0.7. The E_. c o l i s t r a i n C600 r, ntt- su* (2) was used fc12 k 1 2 II f o r c l o n i n g the T7 fragments. The b a c t e r i a l s t r a i n c a r r y i n g the plasmid pBR322 was from the l a b o r a t o r y of H. Boyer. P u r i f i e d pBR322 DNA was used to transform C600 and the r e s u l t i n g s t r a i n designated C600/pBR322. B a c t e r i a l s t r a i n s c a r r y i n g plasmids w i t h T7 i n s e r t s were designated w i t h the p r e f i x pRS. E_. c o l i s t r a i n s were grown i n L-Broth at 30°C unless otherwise i n d i c a t e d . Plasmid c a r r y i n g s t r a i n s were grown i n L-Broth plus 10 ug T e t r a c y c l i n e (Tc)/ml. Plasmid s t r a i n s were stored on L-Broth s l a n t s plus 10 ug Tc/ml. T7 +, VLG3 and a l l amber phage were o r i g i n a l l y from the Studier c o l l e c t i o n . A map showing the approximate p o s i t i o n of each amber mutation i s given i n F i g . 1. Lambda DNA was i s o l a t e d from the s t r a i n XCI857-S7. 7 Materials Agarose powder and Bio-gel A-5M were purchased from Bio-Rad Laboratories. Sephadex G-100 was purchased from Pharmacia. Tetracyline and Chloramphenicol were obtained from Sigma Chemical Co. Ampicillan was purchased from Ayerst Laboratories. 3 3 [2,8- H]dATP (20 ci/mmole) and [2,8- H]dTTP (20 ci/mmole) were purchased 32 from Amersham-Searle. a[ P] dATP (>300 ci/mmole) was purchased from New England Nuclear of Canada. Enzymes The restr ic t ion endonucleases PstI'1 , Hpal 2 , and Hind I I I 3 were purchased from New England Bio Labs. Restriction endonuclease EcoRl1* and Terminal Deoxynucleotidyl Transferase were purchased from Miles Laboratories. E_. c o l i DNA Polymerase I and T4 DNA Polymerase were gifts from Dr. M. Smith and Dr. C. A s t e l l , respectively. Dr. As te l l prepared the T4 DNA Polymerase. Lysozyme, RNAse, and Pronase were purchased from Worthington. 1 Pst I res tr ic t ion endonuclease was isolated from Providencia s tuar t i i (27). In this thesis the enzyme w i l l be referred.to as Pst I. 2HpaI restr ict ion endonuclease was isolated from Haemophilus parainfluenza (25). In this thesis the enzyme is referred to as Hpa I. 3Hind III res tr ic t ion endonuclease was isolated from Haemophilus influenza R^ (20):. The enzyme w i l l be referred to as Hind III in this thesis. ^EcoRI restr ict ion endonculease was isolated from Escherichia c o l i RY13 (33). The enzyme w i l l be referred to as EcoRI in this thesis. 8 Restriction Endonuclease Digest Conditions EcoRI : 100 mM Tris -HCl pH 7.4, 60 mM NaCl, 8mM MgCl 2 , 100 Ug/ml BSA. The enzyme concentration was 5 units/Ug DNA. Digestion proceeded at 37°C for 2 hours. Pst I : 6 mM Tris-HCl pH 7.4, 50 mM NaCl, 8 mM MgCl 2 , 6 mM 2 -mercaptoethanol, 100 Ug/ml BSA. Enzyme concentration was 0.5 units/ug DNA. 100% digestion was complete in 12 hours at 37°C. Hpa I : 10 mM Tris-HCl pH 7.4, 20 mM KC1, 10 mM MgCl 2 , 1 mM D . T . T . , 100 Ug/ml gelatin. Digests proceeded with an enzyme concentration of 0.2 units/ug DNA, for 15 hours at 37°C. Hind III : 6 mM Tris-HCl pH 7.4, 50 mM NaCl, 6 mM MgC]^  , 100 ug/ml BSA. 1 unit/ug DNA was used in a digestion at 37°C for 2 hours. Preparation of Phage DNA Phage DNA to be used for cloning was isolated by phenol extraction of phage that previously had been purified by banding in CsCl density gradients. Phenol was removed by extensive ether washing and dia lys is against 10 mM Tris-HCl pH 7.5, 1 mM EDTA. Concentration of DNA solutions was carried out by dialys is against solid sucrose. T 7 + and VLG3 DNA used to prepare Southern f i l t e r s was isolated from phage that had been purified through a 0.8 cm x 30 cm Bio-gel A-5M column. 9 Isolation and Purif ication of Plasmid DNAs E_. c o l i strains carrying plasmid DNA's were grown at 30°C in LB 9 plus 10 ug Tc/ml to a density of 10 bacteria/ml. Chloramphenicol was added to a f ina l concentration of-200. JJg/ml, and the culture grown 12 hours at 30°C. Cells were harvested by centrifugation in a Beckman Type 15 rotor at 8000 rpm for 20 minutes. Cells were lysed by the lyso-zyme-triton method (15). Cleared lysates were sedimented in CsCl-Ethidium .Bromide gradients at 33,000 rpm for 72 hours. DNA was removed from the gradient tube by piercing the side of the centrifuge tube with a syringe and withdrawing the solution containing the UV-vlsable DNA band. Ethidium bromide was removed by extracting the DNA with n-butanol. The DNA was dialysed against 20 mM Tris -HCl pH 7.5, 20 mM NaCl, 1 mM EDTA for 6 hours to remove the CsCl. Dialyzed DNA was precipitated in 2 volumes of ethanol 12 hours at - 2 0 ° C , dried in vacuo and resuspended in 10 mM Tris pH 7.4, 1 mM EDTA. Concentrations were determined by spectral analysis. Extension of Linear pBR322 and T7 Hpa I Fragments After digestion with Pst I , l inear pBR322 was precipitated in ethanol dried in vacuo, and resuspended in 100 mM K-cacodylate pH 7.0; 15 mM KH„P0,; 8 mM MgC12; 1 mM DTT; 3 mM dATP. This solution was transferred I 4 3 to the reaction tube containing 2 uc i of a[ H]dATP. Enough Terminal Transferase was added to yield an extension of 40-50 nucleotides per 3' OH end in 30 minutes at 37°C as determined by TCA preciptable radio-act iv i ty . The reaction was halted by freeze-thawing in dry ice-ethanol. Hpa I digested T7 DNA was precipitated in ethanol, dried in vacuo, 10 and resuspended in 100 mM K-cacodylate pH 7.0; 30 mM Tris -HCl pH 7.0; 15 mM KH 2P0 4 1 mM C o C l 2 ; 1 mM DTT, 3 mM dTTP. 2 yc i of a[3H]dTTP and Terminal Transferase was added to a, concentration of 0.5 units/yg DNA. The reaction proceeded for 30 minutes to yield an extension of 40-50 nucleotides per 3' OH at 37°C and was halted by freeze-thawing in ethanol-dry ice. Annealing of Extended pBR322 and T7 DNAs The two extended species of DNA were co-precipitated in ethanol 12 hours at - 2 0 ° C , dried in vacuo and resuspended in 0.1 M NaCl 10 mM Tris pH 7.4, 0.5 mM EDTA. This mixture was boiled 5 minutes then incubated at 70°C for 30 minutes. The solution was diluted 1:10 with 70°C annealing buffer then slow-cooled to 37°C and held at this temperature for 6 hours. After the 6 hour incubation the DNA solution was cooled to room temperature and placed on ice. CaCl 2 was added to a f ina l concentration of 0.03 M. Transformation with Annealed PBR322/T7 DNA E_. c o l i C600 r, UL" su* was grown to 10^ bacteria/ml at 37°C in R12 k12 1 1 LB. Cells were treated with NaCl and CaCl 2 according to Cohen et. a l . (5). The prepared ce l l s were incubated with DNA for one hour at 0°C, 3 minutes at 42°C, and placed on ice for an additional hour. The mixture was diluted 1:10 in LB and grown for three generations at 30°C. 0.1 ml samples were plated on LB plates containing 10 yg Tc/ml and grown 12 hours at 37°C. 11 Preparation of Nitrocellulose Membranes Containing the DNA from  Transformed Colonies or DNA Electrophoresed on Gels Transformed colonies were transferred to s ter i le nitrocel lulose (NC) membranes. These membranes were placed on LB plates and incubated at 30°C for 12 hours. Then, the DNA in the colonies was denatured and fixed to the membrane by the method of Grunstein and Hogness (12). The prepared membranes were dried iii vacuo and used for hybridizations with 32 P DNA's. Electrophoresed DNA's were transferred to NC membranes by the method of Southern (28). Nick Translation of DNAs Nick Translation (18) reactions were carried out in 50 mM Tris -HCl pH 7.4; 5 mM MgCl 2 ; 10 mM 2-mercaptoethanol; 50 yg/ml BSA; 50 yM dTTP, 32 dCTP, and dGTP; and 2 yc i of aP-dATP. The reaction was started by the addition of DNA polymerase I from E_. c o l i . The mixture was incubated for 2 hours at 25°C. The reaction was stopped by the addition of EDTA to a f ina l concentration of 20 mM. The DNA was extracted with one volume of phenol, ether washed, and placed on a Sephadex G-100 column made from a 1.0 ml plastic pipette purchased from Falcon Industries. The DNA was eluted with 20 mM Tris-HCl pH 7.5, 1 mM EDTA buffer. A sample was 32 precipitated in TCA to determine the amount of incorporated a P-dATP. 32 Hybridization of P-T7 DNA to Hogness or Southern F i l t ers Hybridization of radioactive DNA was carried out in PM buffer (0.2 mg/ml F i c o l l , 0.2 mg/ml Polyvinylpyrolidone, 0.2 mg/ml BSA, 0.01 mg/ml thymidine, 0.1% SDS and 3xSSC). F i l t ers were incubated in PM at 65°C for 6 hours prior to hybridization. Labeled DNA's were boiled 5 minutes and added to the pre-incubated f i l t e r s . Hybridizations proceeded 15 hours at 65°C. Hybridized fi lters.were extensively washed in 2x SSC, dried in vacuo, and exposed to Kodak X-Omat R fi lm. Marker Rescue Tests Plasmid containing bacteria were grown from a 1:100 di lut ion of a fresh overnight culture in LB to 3 x 10^ cel ls /ml at 30°C. Infecting amber phage (MOI = 5) were added to 1.0 ml of the culture, and incubation was continued. At 6 minutes post-infection infective centers and survivors were plated through T7 antisera on the permissive bacteria 0-11' Also at 6 minutes, the infected ce l l s were diluted 1:100 into fresh LB and grown 70 minutes. Progeny phage were t i tred on permissive and non-permissive hosts to measure the burst size and rescue frequency. The marker ..rescue frequency was calculated as the number of progeny on the non permissive host divided by the number of phage on 0-11'. The frequency of resuce from the T7 plasmid carrying strain was normalized with divis ion by the frequency found in C600/pBR322. A marker rescue test on plates was developed as a fast screening method for genetic markers. Plating efficiency could not be used as a screening procedure, because the T7 plasmids were carried in s u + ce l l s . In this "spot test", LB plates were seeded with a lawn of non-permissive bacteria. 2 y l of plasmid carrying bacteria of various dilutions were over-spotted with 2 y l of amber phage. When plaques arose from spots containing hybrid plasmid carrying ce l l s and no plaques appeared on spots of the same di lut ion of C600/pBR322, the test was considered positive. 13 Preparation.of mRNA Fi l t ers and Hybridization mRNA f i l t e r s were prepared in order to determine the messenger sequences encoded on the inserted T7 fragments. Early T 7 + messenger RNA was isolated from cel ls at 6 minutes post-infection by the method of Young and Pachl (34). The purified RNA was electrophoressed on 1.5% Agarose gels for two hours at 100 V in 10 mM phosphate buffer pH 7.0. Gels were treated with 60 mM NaOH for thirty minutes then neutralized with 200 mM sodium phosphate buffer pH 7.0 for thirty minutes. Just prior to reaction with single stranded RNA's, ABM paper was converted to the diazobenzyloxymethyl (DBM) form by the method of Alwine et. a l . (1). Electrophoretically separated, denatured RNA's were transferred to the DBM paper in a manner analogous to Southern^type transfers. After transfer, f i l t e r s were dried at 80°C in vacuo. Radioactive plasmid DNA's were hybridized to the mRNA f i l t e r s in 50% Formamide/PM buffer at 42°C for 48 hours. After hybridization the f i l t e r s were washed extensively in cold 2x SSC and dried in vacuo before autoradiography. Electroelution of DNA from Agarose Gels Portions of agarose gels containing desired DNA fragments were homogenized in a Pyrex #7725 tissue grinder containing 1.0 ml of elution buffer; 200 mM Tris-Acetate pH 8.0, 10 mM EDTA. The homogenate was eluted through a ver t ica l 5 ml plast ic pipette into a dia lys is bag by applying 150 volts per tube for 8 hours. The eluted DNA was phenol extracted ABM paper was prepared by treating Whatman 540 paper with l - [m-nitro-benzyloxy)methyl] Pyridinium Chloride according to reference 1. 14 ether washed, and dial ized against 10 mM Tris-HCl pH 8.0; 1 mM EDTA, ethanol precipitated, dried in vacuo, and resuspended in enzyme buffer. The purity of an eluted fragment could be established by electrophoresis on an agarose gel. Molecular Weight Analysis Molecular weights were determined by agarose gel electrophoresis of EcoRI digested plasmid DNA's. Since pBR322 DNA has only one EcoRI res tr ic t ion site and T7 DNA has none, a l l hybrid plasmids were converted to linear molecules upon digestion with EcoRI. Hind III digested lambda DNA fragments were used as known molecular weight markers. Linear regression analysis, u t i l i z i n g an HP9810 A calculator, was performed in order to determine the molecular weights. Containment A l l procedures were carried out under A-M physical containment conditions as specified by the Medical Research Council of Canada. 15 RESULTS Principle of the Procedure pBR322 was cut with Pst I to yield a population of l inear molecules. These were extended with single strand poly A segments using Terminal Transferase. T'7 DNA was digested with Hpa I to yield 19 blunt ended fragments. T7 fragments were extended with poly T segments. The two DNA species were annealed and the resulting hybrid molecules were used - + + to transform E . c o l i C600 r m, s u T T " Transformants were plated on 12 12 LB plates containing Tc then repl ica plated to LB plates containing Ap. Colonies which grew on Tc and did not grow on Ap were transferred to NC membranes for hybridization procedures. These colony f i l t e r s were 32 hybridized with P-T7 phage DNA. The colonies which hybridized T7 DNA were characterized by (i) molecular weight analysis ( i i ) radioactive plasmid DNA - "Southern" f i l t e r hybridizations ( i i i ) genetic analysis by marker rescue experiments. I. Construction of Recombinant Plasmid Molecules  Digestion of pBR322 DNA 5'..CTGCA+G..3' The R.E . Pst I has the recognition sequence 3'. .G+ACGTC..5'. Complete digestion of pBR322 with this enzyme yields a population of l inear molecules. The cleavage site fa l l s near the beginning of the gene coding for ampicillan resistance (Fig. 2). Linear molecules of pBR322 have extended 3' OH ends. Digested pBR322 can be visualized on 0.5% Agarose The cloning of T7 DNA fragments is outlined in Figure 3. 3'0H-AAAp pBR322 T 7 RE Pst I RE Hpa I V 19 Fragments •3'0H Terminal Transferase pAAA Terminal Transferase TTTp" .pTTT Anneal & Transform Figure 3. Outline of Cloning Procedure gels stained with ethidium bromide (Fig. 4). The l inear molecules migrate between the bands representing open c i rc l e and supercoiled forms of the DNA. Purified plasmid DNA was ethanol precipitated, sedimented by centrifugation, dried in vacuo, and resuspended in Pst I digest buffer. The reaction was started by the addition of enough Pst I to yield 100% linear molecules in 12 hours at 37°C. The reaction was terminated by freeze-thaw in ethanol-dry ice. The digested DNA was precipitated in ethanol, centrifuged, dried in.vacuo, and resuspended in 10 mM Tris-HCl pH 7.4, 1 mM EDTA. Digestion of T 7 + and VLG3 DNA Purified DNA from wild type T7 phage or the deletion mutant LG3 was precipitated in ethanol, centrifuged, and resuspended in Hpa I digest buffer. Restriction endonuclease Hpa I, which has the recognition sequence GTT+AAC, was added to start the reaction which proceeded 15 hours at 37°C. Nineteen fragments with blunt ends (eg. GTT-OHfpAAC) result from this digestion. Samples of the digested DNA were electro-phoresed on 1.6% agarose gels to separate the 19 fragments and determine the extent of digestion. Two bands, I and J, co-migrate on this gel system (Fig. 5). EDTA was added to the digested DNA at a f ina l concentration of 50 mM. This mixture was frozen in dry ice-ethanol and thawed at room temperature twice, ethanol precipitated, sedimented by centrifugation, dried in vacuo and resuspended in 10 mM Tris -HCl pH 7.4; 1 mM EDTA. A B C Figure 4. A n a l y s i s of pBR322 DNA on a 0.5% agarose g e l . E l e c t r o p h o r e s i s was i n TBE b u f f e r (90 mM T r i s - B o r a t e , pH 8.0, 1 mM EDTA), 3 hours at 7 volts/cm. (A) EcoRI digested pBR322; (B) Pst I digested pBR322; (C) non-digested pBR322. 19 o — '•"CM*** ,y/iM^v* I . ; - ' - t Figure 5. Hpa I restr ict ion analysis of (A) T7 DNA and (B) VLG3 DNA. Electrophoresis was carried out on a 1.6% agarose gel in 50 mM phosphate buffer pH 7.0. Gels were routinely run at 3 volts/cm for 6 hours. (Adopted from Studier, ref. 19). 20 Extension of DNA's with, homopolymer blocks The addition of homopolymer segments of deoxynucleotides to double stranded DNA for the purpose of joining two DNA molecules was f i r s t reported by Loban. and Kaiser (17). The additon of mononucleotides to the 3' OH termini of DNA is catalyzed by the enzyme Terminal Deoxy-nucleotidyl Transferase. The extended 3' OH ends of Pst I digested pBR322 DNA provide an ideal substrate for this enzyme, thereby eliminating a X exonuclease digestion required when no external 3' OH is available. For example, when EcoRI digested DNA is used as the substrate, mononucleotides must be removed from the 5'phosphoryl termini to expose the single stranded 3' OH ends. These extended 3' OH ends are necessary to overcome an i n i t i a l lag in the kinetics of the terminal transferase reaction (17). The extension reaction proceeds from the 3' OH termini of the double stranded DNA by adding nucleotide monophosphates derived from triphosphate molecules. Single stranded homopolymer extensions result from this reaction. If the DNA contains internal discontinuities or nicks, addition of homopolymer strands can occur in these regions. It was necessary to use DNA preparations that were as free of these discontinuities as possible. Another problem that arises in the terminal In this paper, a nick refers to a single, internal break at a phosphodiester bond leaving a free 3' OH and 5' phosphate group. Discontinuity can indicate a nick or larger internal deletion with 3' OH termini. transferase reaction is the "self- init iated" polymerization of the deoxymononucleotides. This was minimized by adding KH^PO^ to the react-ion mixture at a f ina l concentration of 0.15 M (14,11). 3 The addition of a[ H]-dATP. or dTTP to the reaction mixture provided a method of monitoring the number of nucleotides added to each 3' OH end. Samples, usually 5 y l , were removed from the reaction at various times and the cold TCA precipitable radioactivity was measured. Accuracy in determining the number of nucleotides added depends on an exact measurement of the amount of DNA and the precise amount of cold triphosphate used in each reaction. The radioactive triphosphate used was at a high specific act iv i ty so the contribution to the substrate triphosphate concentration was very low. It is not known what percentage of the molecules were extended to the calculated length. By t i t ra t ing the terminal transferase i t was possible to control the reaction to yield the calculated extention length in 30 minutes at 37°C. Several preparations of DNA were overextended (200-300 nucleo-tides/3' OH). It was found that these extensions could be enzymatically degraded to the desired length by u t i l i z i n g the 3' -> 5' exonuclease act iv i ty of T4 DNA polymerase (10). The degradation was monitored by the loss of radioactivity from the DNA. This enzyme w i l l not attack double stranded DNA. Pst I digested, l inear pBR322 was extended in terminal trans-I | ferase conditions where Mg served as the divalent cation. However, under the same conditions, the blunt ended Hpa I fragment showed no nucleotide incorporation. According to Wu et_ a l . (23), the substitution of Co ions for Mg ions allows the rapid extension of blunt ended 22 double stranded DNA's with terminal transferase. When this substitution was made, incorporation of dTMP molecules to the 3' OH ends of the Hpa I 3 fragments proceeded rapidly. The use of [ H]-dTTP to monitor incorporation of deoxynucleotides allowed computation of the average number of bases added. A n n e a l i n g Once the T7 fragments and the pBR322 DNA were extended, they were co-precipitated in ethanol, centrifuged, dried in vacuo, and resuspended in annealing buffer at a concentration of 30-40 Ug / m l . The rat io of T7 DNA to pBR322 in this mixture was 2:1. This solution was boiled 5 minutes then incubated at 70°C for 30 minutes to allow i n i t i a l hybridization of the plasmid DNA strands. The concentration was reduced 10-fold by addition of 70°C annealing buffer and the temperature reduced slowly to 37°C. The annealing of the poly A- poly T extensions occurs during this cooling period. The exact temperature at which these A-T regions anneal depends on the length of overlap. The low concentration (3-4 ug /ml) fac i l i ta tes the annealing of the poly A- poly T regions (22). The annealed solution was stored at 4°C u n t i l used for trans-formation. It was important to remember that this DNA was not repaired and large gaps existed which increased i ts l a b i l i t y to temperature changes. The temperature of disassociation (Tm) for a region of poly A -T, 40 bases in length, is approximately 35° - 40°C in 0.1 M NaCl, as suggested by denaturation studies on sythetic oligonucleotides (personal communication, M. Smith). No attempt was made to repair these molecules in v i t r o . Instead, the non-repaired c irc les were used to transform c ol i> thereby allowing the bacterial repair system to complete the 23 joining process. Transformation After treatment with 0.3 M CaCl^, the prepared ce l l s were mixed with the annealed DNA and incubated at 0° C for 60 minutes. The suspension was heat pulsed 3 minutes at 42°C and placed at 0°C for another 60 minutes. The second hour at 0°C increases the frequency of transformation (22). A typical transformation frequency for experiments using 2 hybrid, non-repaired plasmid DNA was 4 x 10 transformants per ug of pBR322 DNA. Control transformations with pBR322 and cut-extended pBR322 4 yielded frequencies of 10 transformants/ug pBR322 and 0 transformants/ Ug pBR322 respectively. The number of transformed ce l l s decreased when the length of the poly A - T extensions exceeded 50 nucleotides per 3' OH end (Table 2). II. Selection and Characterization of Plasmids Carrying T7 DNA After transformation, ce l l s were grown for 2-3 generations in LB, then concentrated 10-fold and plated on LB plates containing 10 ug Tc/ml. The plates were incubated at 37°C for 12 hours. Cel ls then were repl ica plated to LB plates containing 100 yg Ap/ml. 98% of the cel ls S R were found to be Ap and Tc . These transformants were picked and spotted on NC membranes. The membranes were prepared by the method of Hogness 32 + and Grunstein (12). The f i l t e r s were hybridized with a P - T7 phage DNA probe prepared by nick translation. Approximately 70% of the original S R 32 Ap , Tc clones hybridized the P-DNA indicating the presence of T7 DNA within these clones. T 7 + DNA does not hybridize to E_. c o l i or pBR322 DNA alone (Fig. 6). TABLE II TRANSFORMATIONS FREQUENCIES WITH VARYING POLY (A)-(T) CONNECTOR LENGTHS Poly A-T length Transformation frequency (CFU/ug pBR322) 40/3' OH 400 150/3' OH 60 > 300/3' OH none detectable 25 Figure 6. Colony Hybridization: N i t r o c e l l u l o s e f i l t e r s contain denatured S R DNA from transformants selected on the basis of Ap and Tc . + 32 T7 DNA was made radioactive by nick t r a n s l a t i o n with a - P-dATP. Hybridization was c a r r i e d out at 65°C for 15 hours. The colonies which appear dark on the autoradiogram contain T7 DNA. The f i l t e r s at the l e f t were exposed 5 days and those on the r i g h t for 2 days. The round f i l t e r at the far right contains DNA from pSR322/C . colonies. Identification of a clone containing Hpa. I fragment E Eight clones, which were found to contain T7 DNA by hybrid-32 + ization with P - T7 DNA, were picked at random and cleared lysates were prepared. Purified plasmid DNA was made radioactive by nick translation. The radioactive DNA's were hybridized to Southern f i l t e r s containing the DNA from a T7/Hpa I digest. One of these clones, pRS142, was identified as carrying Hpa I fragment E (Fig. 7). Single step experiments were done to determine the marker rescue frequencies for the various markers located on this T7 fragment. Table 3 shows the frequencies and the normalized frequencies for these experiments. Control experiments showed no recombination for markers outside the Hpa I E fragment. The clone PRS142 was used to establish the conditions for the spot marker rescue tests. Figure 8 shows the result of such a test. Tests of this type were used to confirm genetic markers in other clones and aid in their ident i f icat ion. The cloning of part ia l Hpa I D fragments 2 ug of- Hpa I fragment D was removed from an agarose gel by 32 electroelution. The purified fragment was nick translated with a P-dATP and hybridized to Hogness f i l t e r s containing 230 of the original transformants known to contain T7 DNA. Figure 9 shows that 15 clones hybridized the radioactive fragment. Four of these clones, pRS88, 94, 202, 229 were chosen at random and purified plasmid DNA's were prepared. The molecular weights of these plasmids were determined by agarose gel electrophoresis (Fig. 10). Spot marker rescue tests revealed that none of the four clones rescued both 5am28 and 4am208. pRS88 was positive 27 ;  A B Figure 7. Hybridizaton of radioactive DNA's to n i t r o c e l l u l o s e f i l t e r s prepared by the method of Southern (28) . The A and B f i l t e r s contain Hpa I digest VLG3 DNA. F i l t e r s C-F contain Hpa I digested T 7 + DNA. Hybridization was with the following labeled DNA's; (A) T7 +; (B) pRS148; (C) T7 +; (D) pRS136; (E) pRS142; (F) pRS29. 28 TABLE I I I RESCUE FREQUENCY OF AMBER MUTANTS BY pRS142 AND pBR322/C 6 0 0 Rescue Frequency Normalized Amber pBR322/CftQO pRS142 Frequency 12 am 3 1 x 1 0 _ 4 1.1 x 10~ 4 1.10 12 am 65 1.2 x 10~ 4 1.3 x 10~ 4 1.10 13 am 149 4 x 10~ 4 9.3 x 10~ 2 2.26 x 10 2 13 am 89 1.2 x 10~ 4 1.8 x 1 0 _ 1 1.5 x 10 3 14 am 140 2.3 x 1 0 _ 6 1.16x 1 0 _ 2 7.8 x 10 3 14 am 35 1 . 6 5 x l 0 ~ 4 5.2 x l O _ 2 3.2 x l O 2 15 am 31 2.6 x 10~ 4 2.65xl0~ 4 1.02 Frequency of marker rescue during single step recombination experiments performed on pRS142 and pBR322/C Normalized frequency refers to the frequency of rescue for pRS142 divided by the frequency for pBR322/C f i n f ). 29 Figure 8. Recombination spot test of pRS142. Plates were seeded with non-permissive bacteria and 2 ul of 14aml40 were spotted at the dilutions indicated. 2 ul of plasmid containing ce l l s are over-spotted at the dilutions shown. A positive test is explained in the methods section of this paper. A m -5 A m -6 A m -7 I 1 I 31 Figure 9. Colony hybridization of 230 clones with P-Hpa I restr ict ion fragment. The Hpa I fragment D was isolated from a 1.6% 32 agarose gel and nick translated with a P-dATP. F i l t ers were hybridized at 65°C for 15 hours. M.W. X10 6.4 2.9 1.6 1.4 A B C D E CI H I J K Figure 10. Molecular weight analysis of recombinant plasmids on 0.8% agarose gels electrophoresed in TBE buffer for 15 hours at 12 mA. (A,F) Hind III cut A DNA. A l l other plasmids were digested with EcoRI; (B) PRS148; (C)pRS134F; (D) pRS142E; (E,K) PBR322; (G) PRS88; (H) pRS94; (I) pRS202; (J) pRS229. for 4am208 and pRS94, 202 and 229 were positive for 5am28. This finding was not unexpected for the molecular weights of these clones were found to be much less than the expected value i f they had carried the entire D fragment. Therefore a l l the clones screened were found to be carrying part ia l D fragments. Extensive recombination tests with a greater number of genetic markers w i l l be needed to establish the extent and position of these fragments in relation to the genetic map. pRS202 was further characterized as carrying gene 5 by hybrid arrest trans-lat ion (21). Cloning the origin of replication - Hpa I fragment G Among the eight clones chosen at random one clone, pRS202 was found to contain either Hpa I fragment G or H. The ambiguity arose from the fact that these two fragments migrate on an agarose gel very close together due to the small difference in their molecular weights. 32 Therefore, a Southern f i l t e r hybridized with P-pRS136 was not a definite ident i f icat ion. A mRNA f i l t e r was prepared containing early 32 T7 messenger RNA's (22). P labeled pRS136 was hybridized to this f i l t e r in 50% formamide-PM buffer at 42°C for 48 hours. The auto-radiogram shows hybridization with the 1.0 and 1.3 mRNA bands (Fig. 11). This corresponds to the Hpa I fragment G. The molecular weight of the hybrid plasmid is correct for fragment G. Genetic analysis by spot test showed that pRS136 rescued lam342a and 1.3amHal3. Therefore, this This work was carried out with the assistance of Susan Strome at the University of Washington, Department of Biochemistry and Genetics, Seattle, Washington. Figure 11. Hybridization of P-DNA to early T7 mRNA f i l t e r s . (A) wild type T7 DNA labeled by nick translation (B) pRS136 labeled by nick translation. plasmid carried the major origin of replication of T7 DNA, which l i e s between genes 1.0 and 1.3 (8). Additional clones carrying T7 genes pRS148 was i n i t i a l l y characterized as carrying either Hpa I fragments I or J by hybridization of nick translated plasmid to a Southern f i l t e r containing a Hpa I/VLG3 digest (Fig. 7). Bands I and J co-migrate in this gel system. Genetic analysis was attempted and i t was found that pRS148 would not plate wild-type T7 phage nor any amber phage. No marker rescue analysis could be carried out to determine genetic markers. Upon infection with wild-type phage, no wild-type progeny are l iberated. This phenomenon w i l l be studied in deta i l in future experiments. Identification of this fragment w i l l be possible u t i l i z i n g hybrid-arrest translation or by hybridization with radio-active Hpa I fragment J labeled by T4 polynucleotide kinase. pRS3 and pRS29 were identified as sub-fragments of Hpa I fragment A. Figure 7 shows an autoradiogram of radioactive pRS29 hybridized to a Hpa I/T7 Southern f i l t e r . Genetic analysis by spot test shows that pRS3 carries the marker 10aml3 and llam37, while pRS29 carries only llam37. The molecular weights of these inserts were approximately 1.3 x 10 daltons. pRS29 must extend to the right into gene 9. Hybrid arrest translation results confirmed these results. 37 DISCUSSION Hpa I res tr ic t ion fragments of bacteriophage T7 DNA have been inserted into plasmids in order to fac i l i ta te the biochemical and genetic analysis of this phage. The work presented here i l lus trates the process of constructing these hybrid molecules and their characterization. We have used Hpa I fragments because they have previously been mapped. This makes i t possible to select specific regions of interest on the T7 genome and study their behavior in a plasmid system. The fragments were inserted by the poly A - poly T connector method. The use of poly G-C ta i l s for insertion, reconstructs the Pst I s i te of pBR322 which would fac i l i ta te the removal of the T7 inserts. For.our purposes the G-C connectors were not necessary. Further cloning of sub-fragments w i l l u t i l i z e this method i f large amounts of DNA are required. With the poly A - T extensions i t is possible to selectively heat denature the A-T regions then excise the fragments by digestion with Si nuclease (13). The original intent of this work was to clone specific fragments of T7 DNA. One of these was the Hpa I fragment A. 10 ug of fragment A were isolated from a preparative gel and extended to d(A)^^ per 3' OH end by Terminal Transferase. No transformants were found with this preparation. This is in agreement with Studier's information that fragment A was not found in any clones from shotgun-type experiments (4). Several clones contained sub-fragments of Hpail fragment A. pRS3 and pRS29 are examples of this type of sub fragment clone. It is not known why a l l of fragment A has not been cloned intact. It was decided to screen clones from a "shotgun" type experiment 38 because the extension of single fragments isolated from gels was not accurate. The exact amount of DNA isolated from a gel could not be determined. Therefore, the calculation of extension length was only an estimate. Also, the small amount of DNA isolated from gels made control of the extension reaction d i f f i c u l t . Transformation of C ^ Q Q using total T7 DNA cut with Hpa I and extended with terminal transferase was carried out. This shotgun approach yielded ample transformants which were probed using the various methods described in this paper. The use of radioactive single Hpa I fragments as probes was a very effective method of detecting clones carrying a specific insert . Hybridization of a Hpa I fragment did not necessarily indicate the presence of the entire fragment within a clone. Genetic analysis indicates the extent of inserts as does molecular weight determination. A rapid in vivo;marker rescue screening procedure was developed to obtain i n i t i a l genetic marker information on the recombinant plasmids. When rescue frequencies were desired, single-step growth experiments were performed. One of the interesting properties of these plasmids is their a b i l i t y to recombine with phage DNA. This presents a method for intro-ducing mutations into the T7 genome via recombination, and a method for refining the genetic map of T7 with marker rescue frequency data. The biological properties of the T7 plasmids constructed in this work, i e . . t ranscr ipt ion , translation, recombination, and their use as mutagens w i l l be the subject of future investigations. In summary, Hpa I res tr ic t ion fragments of wild type T7 DNA have been inserted into the plasmid pBR322. Two hundred f i f t y clones 32 hybridizing P-T7 DNA were isolated. Clones carrying Hpa I fragments G and E were characterized by h y b r i d i z i n g P-plasmid DNA to Southern-type f i l t e r s containing electrophoresed Hpa I/T7 fragments. Genetic analysis by marker rescue was performed and the r e s u l t s confirm the i d e n t i t y of these two clones. Several clones were selected by hybrid-i z a t i o n with ra d i o a c t i v e s i n g l e Hpa I fragment D. These clones were further i d e n t i f i e d by marker rescue data which indicated the presence of T7 genes 4 and 5. Two clones that c a r r i e d sub fragments of Hpa I fragment A were i d e n t i f i e d by h y b r i d i z a t i o n and genetic studies. The methods described i n t h i s paper should allow the c h a r a c t e r i z a t i o n of any hybrid plasmid carrying T7-Hpa I fragments. 40 BIBLIOGRAPHY .1. Alwine, J . C . , Kemp, D . J . , Stark, G.R. (1977) Method for detection of specific RNA's in agarose gels by transfer to diazobenzyl-oxymethyl-paper and hybridization with DNA probes. Proc. Nat l . Acad. Sc i . U.S.A. 74:5350-5354. 2. Bachman, B . J . (1972) Pedigrees of some mutant strains of E_. c o l i K l 2 . Bacteriol . Rev. 36:525-557. 3. Bolivar, P . , Rodriguez, R. , Greene, P . , Bettach, M . , Heynecker, H . , Boyer, H. (1977) "Construction and characterization of new cloning vehicles II . A multiple" cloning A system. Gene 2_:95-113. 4. Campbell, J . , Richardson, C , Studier, F.W. (1978) Genetic recomb-ination and complimentation between bacteriophage T7 and cloned fragments of T7 DNA. Proc. Natl . Acad. Sc i . U.S.A. 75:2276-2280. 5. Cohen, S.N. , Chang, A.C.Y. . , Hsu, L . (1972) Non-chromosomal an t i -b iot ic resistance in bacteria: Transformation of E_. c o l i by R-factor DNA. Proc. Natl . Acad. Sc i . U.S.A. 69:2110-2114. 6. Davis, R.W., Hyman, R.W. (1970) Physical locations of the in v i tro RNA in i t ia t ion site and termination sites of T7 DNA. Cold Spring Harbor Symp. Quant. B i o l . 35:269-282. 7. Delbriick, M. (1946) Bacterial viruses or bacteriophages. B i o l . Rev. 2JL:30-41. 8. Dressier, D . , Wolfson, J . , Magazin, M. (1972) Ini t iat ion and re-i n i t i a t i o n of DNA synthesis during replication of Bacteriophage T7. Proc. Natl . Acad. Sc i . U.S.A. 69^:998-1002. 9. Dunn, J . J . , Studier, F.W. (1975) Effect of RNAse III cleavage on Translation of Bacteriophage T7 messenger RNA's. J . Mol. B i o l . 97_:487-499. 10. Englund, P.T. (1971) Analysis of nucleotide sequences at 3' termini of duplex DNA with the use of T4 DNA polymerase. J . B i o l . Chem. 246:3269-3276. 11. Glover, D.M. (1976) The construction and cloning of hybrid DNA molecules. New Techniques in Biophysics and Ce l l Biology, pp. 124-145. 12. Grunstein, M . , Hogness, D. (1975) Colony Hybridization: A method for isolat ion of cloned DNA's that contain a specific gene. Proc. Nat l . Acad. Sc i . U.S.A. 72:3961-3965. 41 13. Hofstetter, H. , Schambock, A..., Van den Berg, J . , Weissman, C. (1976) Specific excision of,the inserted DNA segment from hybrid plasmids constructed by the poly(dA) - poly(dT) method. 14. Kato, K - I . , Goncalves, J . Houts, G.E. and Bollum, F . J . (1967) Deoxynucleotide-polymerizihg enzymes of calf—thymus gland. II . Properties of the terminal, deoxynucleotidyl transferase. J . B i o l . Chem. 242:2780-2789. 15. Katz, L . , Kingsburry, D , Helinsky, D. (1973) Stimulation by cAMP of plasmid DNA replicat ion and catabolite repression of plasmid DNA-protein relaxation complex. J . Bacteriol . 114:477-591. 16. Ken, C , Sadowski, P.O. (1974) Packaging and maturation of DNA of Bacteriophage T7 in v i t r o . Proc. Natl . Acad. Sc i . U.S.A. 7^1:3545-3549. 17. Loban, P . , Kaiser, A. (1973) Enzymatic end to end joining of DNA molecules. J . Mol. B i o l . 78:453-471. 18. Maniatis, T . , Jeffery, A . , Kle id , D. (1975). Nucleotide sequence of the rightward promoter of Phage X. Proc. Natl . Acad. Sc i . U.S.A. 72_: 1184-1188. 19. McDonnel, M . , Simon, M . , Studier, F.W. (1977) Analysis of Restr ict -ion Fragments of T7 DNA and determination of Molecular Weights by Electrophoresis in neutral and alkaline gels. J . Mol. B i o l . 110:119-146. 20. Old, R. , Murray, K . , Roizes, G. (1975) Recognition sequence of res tr ic t ion endonuclease III from Haemophilus influenza. J . Mol. B i o l . 92:331-339. 21. Patterson, B . M . , Roberts, B . E . , Kuff, E . L . (1977). Structural gene identif icat ion and mapping by DNA - mRNA hybrid-arrested ce l l - free translation. Proc. Natl . Acad. Sc i . U.S.A. 74: 4370-4374. 22. Rabbitts, T .H. (1976) Bacterial cloning of plasmids carrying copies of rabbit globin messenger RNA. Nature 260:221-225. 23. Roychouldhuvy, R. , Wu, R. (1976) Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by Terminal deoxy-nucleotidyl transferase. Nuc. Ac. Res. 3_:863-877. 24. Sadowski, P .D . , Vetter, D. (1976) Genetic recombination of Bacteriophage T7 DNA iri v i t r o . Proc. Nat l . Acad. Sci . U.S.A. 73:692-696. 42 25. Sharp, P . , Sudgen, B . , Sambrook, . J . (1973) Detection of two res tr ic t ion endonuclease act iv i t ies in Haemophilus parainfluenza using analytical agarose ethidium bromide electrophoresis. Biochemistry 12_: 3055-3063. 26. Sinsheimer, R .L . (1977) Recombinant DNA. Ann. Rev. Biochem. 46:415-438. 27. Smith, Blatter Davies. (1976) Nuc. Acid Res. 3:343. 28. Southern, E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J . Mol. B i o l . 98:503-517. 29. Studier, F.W. (1969) The genetic and physiology of Bacteriophage T7. Virology 39:562-574. 30. Studier, F.W. Bacteriophage T7. Science 176:367.-376. 31. Studier, F.W. (1973) Genetic analysis of non-essential bacteriophage T7 genes. J . Mol. B i o l . 79:227-236. 32. Studier, F.W. (1975) Gene expression after bacteriophage T7 infection. Proc. of the 10th FEBS Meeting, pp. 45-53. 33. Yoshimori, R.N. (1971) Ph.D. Thesis, University of Cal i fornia at San Francisco, Medical Center. 34- Young, E . T . , Pachl, C.A. (1978) The size and messenger RNA act iv i ty of Bacteriophage T7 late transcripts synthesized in vivo. J . Mol. B i o l . 122:69-101. 

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