UNIT I - Molecular Tools for Gene Cloning PDF

Department of Biotechnology, Dayananda Sagar College of Engineering UNIT-1 MOLECULAR TOOLS FOR GENE CLONING Topics: 1.1 Extrachromosomal Elements Gene cloning strategy: Steps in creating a recombinant...

Department of Biotechnology, Dayananda Sagar College of Engineering UNIT-1 MOLECULAR TOOLS FOR GENE CLONING Topics: 1.1 Extrachromosomal Elements Gene cloning strategy: Steps in creating a recombinant 1.2 DNA  Vectors in rDNA technology  Types of vectors: Plasmids, Cosmids, Phagemids 1.3 Viruses  Construction of vectors (Blue script, BAC, YAC, MAC).  Enzymes in rDNA Technology: Nucleases, Restriction Enzymes, RNases, Methylases, Polymerases, Reverse Transcriptase, 1.4  End Modifying Enzymes: Terminal Transferase, T4 Polynucleotide Kinase, Alkaline Phosphatases, linkers and adapters. Genetic Engineering & Applications (22BT52) 1 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering UNIT I MOLECULAR TOOLS FOR GENE CLONING 1.1.Extra chromosomal Elements In addition to the chromosome, many bacteria possess smaller, independently replicating (extrachromosomal) nucleic acid molecules termed plasmids and bacteriophages. Plasmids: Plasmids are independent, self-replicating, circular units of dsDNA, some of which are relatively large (60-120 kb) while others are quite small (1.5-15 kb). Bacteriophages: Bacteriophages are bacterial viruses that can survive outside as well as inside the bacterial cell. Bacteriophages differ from plasmids in that their reproduction usually leads to destruction of the bacterial cell. In general, bacteriophage consists of a protein coat or head (capsid) which surrounds nucleic acid which may be either DNA or RNA but not both. Some bacteriophages may also possess a tail-like structure which aids them in attaching to and infecting their bacterial host. 1.2.Gene Cloning Strategy: Construction of Recombinant DNA Molecules Introduction: Genetic Engineering / Recombinant DNA technology  Genetic engineering is a broad term referring to manipulation of an organisms’ nucleic acid. Organisms whose genes have been artificially altered for a desired affect is often called genetically modified organism (GMO).  Recombinant DNA technology (rDNA) is technology that is used to cut a known DNA sequence from one organism and introduce it into another organism thereby altering the genotype (hence the phenotype) of the recipient.  The process of introducing the foreign gene into another organism (or vector) is also called cloning. Sometimes these two terms are used synonymously.  Chromosomal DNA is not the only genetic material; some bacteria possess extra chromosomal genetic elements called plasmids. Plasmids are circular DNA molecules that can replicate independently.  Plasmids contain the requisite genetic machinery, such as replication origin, which permits their autonomous propagation in a bacterial host or in yeast.  Since plasmids are small stretches of DNA sequences they are easy to handle in vitro and therefore are suitable vectors in genetic engineering.  Foreign DNA sequences can be introduced into bacteria, yeast, viruses, plant and animal cells.  The genes are identified by various methods and once identified; it is convenient to maintain a gene library.  A gene library is a population of organisms, each of which carries a DNA molecule that was inserted into a cloning vector.  Ideally, all of the cloned DNA molecules represent the entire genome of the organism. Basically, these techniques are used to achieve the following:  Study the arrangement, expression and regulation of genes  Modification of genes to obtain a changed protein product  Modification of gene expression either to enhance or suppress a particular product  Making multiple copies of a nucleic acid segment artificially Genetic Engineering & Applications (22BT52) 2 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering  Introduction of genes from organism to another, thus creating a transgenic organism  Creation of organism with desirable or altered characteristics. Isolation of the gene (DNA sequence)  The technique involved in recombinant DNA technology is to slice (cut) the desired DNA segment and introduce it into a vector (e.g., plasmid).  This is achieved using a specific bacterial enzyme called restriction enzymes or restriction endonucleases, which can cleave a DNA sequence at a specific site.  Only those enzymes that cut the DNA asymmetrically are useful in rDNA technology. When such enzymes cleave DNA, they leave single stranded “sticky ends” on both strands.  Same restriction enzymes are used to cleave the DNA molecule to be transferred and the vector.  The circular structure of the plasmid is broken by the restriction enzyme, this process leaves a “sticky end” at either strand. The strand of DNA to be transferred must have two restriction sites; one on either side of the DNA segment of interest. When it is acted upon by restriction enzyme, it generates two sticky ends, one at either side of the segment. Since these sticky ends are generated by the same enzyme, they are complementary and hence are cohesive. Cloning  The sticky ends are generated by the same enzyme on vector as well as the target DNA are complementary and hence are cohesive.  The sticky ends of the cleaved DNA segment cohere with those of the vector, thus the cut DNA sequence can now be introduced into the plasmid.  The cut ends are joined by DNA ligase enzyme and the introduced gene becomes a part of the plasmid.  Ligase is an enzyme that covalently joins the sugar phosphate backbone of bases together.  Ligase will join either "sticky" ends or "blunt" ends, but it is more efficient at closing sticky ends.  The process of introducing foreign gene into a vector is called as cloning and the plasmid containing a cloned gene is called chimera. The DNA sequence that has been inserted into the vector is also called an “insert”.  The chimera is then introduced into its host (e.g., a bacterium) by various methods.  Vectors carrying the genes must be incorporated into the living cells so that they can be expressed or replicated.  The cells receiving the vector are called the host cell and once the vector is successfully incorporated into the host cell, the host cell is said to be “transformed”.  DNA cannot be readily sent across the membrane; following methods like Gene gun, Gold particles coated with DNA, heat shock, Electroporation, Viruse,s Microinjection, or liposome are used. DNA segments are fired into the host cell. Selection of transformed cells  For eg., if pUC18 plasmid containing gene (lacZ’) coding for galactosidase activity is inserted with a foreign DNA. The plasmid also codes for ampicillin resistance.  Due to the insertion, the gene gets interrupted and the bacterium transformed with this plasmid lacks galactosidase activity.  Bacteria lacking this plasmid as well as those transformed by the chimeric plasmid lack galactosidase activity. Genetic Engineering & Applications (22BT52) 3 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering  When grown on medium containing a chromogenic substrate, bacteria containing chimeric plasmid produce colorless colonies.  Bacteria containing plasmid without the insert produce blue colonies and the bacteria not transformed by  Plasmids also produce colorless colonies. If ampicillin is also incorporated in the medium, bacteria not transformed with plasmid do not produce colonies. Thus, on this medium the colorless colonies indicate bacteria that have received chimera plasmid.  Other methods to detect successful transfer of DNA include DNA hybridization and PCR. Fig 1.1: Steps of Gene Cloning Applications of Genetic Engineering  Genetic engineering has wide, applications in modem biotechnology. Since microbial cells have a much higher metabolic rate, genes of desired enzymes could be introduced into plasmid of bacteria.  The bacterial insulin, humulin was prepared by cloning the DNA from chromosome number 11 of human cells in bacteria.  The hormone somatostatin, thymosin alpha-I, as well as Beta-endorphin has been produced by genetically engineered microorganisms. Genetic Engineering & Applications (22BT52) 4 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering  Subunit vaccines can be prepared by cloning the DNA coding for the antigenic protein present on a pathogen. E.g, Hepatitis B, Foot & mouth disease, malaria etc. Plants can be made to express antigenically important microbial proteins (edible vaccine).  Weissmann and his associates have produced alpha-interferon by recombinant DNA methods.  The enzyme urokinase, which is used to dissolve blood clots, has been produced by genetically engineered microorganisms.  Chimeric monoclonal antibodies with human Fc region can be made using this technology.  This technology has been applied to treat some of the genetic diseases (gene therapy).  AAT (alpha-1 antitrypsin), tissue plasminogen activator, factor VIII, antithrombin, erythropoeitin etc are some of the other proteins produced using this technology. Transgenic animals with required characteristics have been created by this technique. 1.3 Vectors in rDNA Technology What is a vector? A vector is a small DNA molecule capable of self-replication and is used as a carrier of DNA fragment inserted into it for cloning. Vector is also called cloning vehicle or cloning DNA. 1. Salient Features of Vectors  It should be able to replicate autonomously, so that it can generate multiple copies of itself along with DNA insert within a single host  Have origin of replication for replication  It should be easily isolated & purified  The vector should be small in size, low mol wt. Ideally it should be less than 10kb in size, because large DNA molecules are broken during purification and present difficulty during manipulation required for gene cloning.  Non-toxic to host cells  It should be easily introduced into host cell  Have space for foreign inserts  The vector should contain suitable marker/markers to permit its detection in the host cell and for selection of transformants, e.g. antibiotic resistance genes.  Easy transformation of host cell  The vector should have unique target sites for many restriction enzymes, so that DNA insert can be integrated without disrupting the essential functions of the vector.  When the objective is gene transfer, the vector should have the ability to integrate either itself or the DNA insert it carries into the genome of the host cell. 2. Types of Vectors: Natural available (modified and then used):  Plasmid vectors-bacterial cell  Bacteriophage vectors.  Viral/viruses vectors. Hybrid/construct vectors:  Phagemid vectors- plasmid +phage  Cosmid vectors. Genetic Engineering & Applications (22BT52) 5 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering  Phasmid vectors.  BACs [Bacterial Artificial Chromosomes].  YACs [Yeast Artificial Chromosomes].  HACs [Human Artificial Chromosomes]. Based on the objective vectors are of two types: Cloning vector: Vector is used only for propagation or cloning DNA insert inside a suitable host cell is called a cloning vector. The cloning vector contains relaxed replication control so that they can produce multiple copies of each transformed cell. Expression vector: A vector is termed as expression vector, when it is used to express the DNA insert producing the specified protein. Such vectors contain the regulatory sequences such as promoters, operators and ribosome binding site (RBS). Expression vectors have prokaryotic promoter and RBS just before the eukaryotic coding sequence because the regulatory sequences of eukaryotes are not recognized in prokaryotes. Vectors broadly belong to two systems: A. Prokaryotic host vector system:  Plasmids  Bacteriophages  Cosmids (artificial constructions) B. Eukaryotic host vector system A. PLASMID VECTORS Definition: Plasmids are extra chromosomal, self-replication, double stranded, circular molecules of DNA found in bacterial cell. Plasmids exist either independently in bacteria or integrated into bacterial chromosome. Generally plasmids are nonessential for bacterial cells except under specific environment. Plasmids contain genetic information for their own replication. They also carry one /more genes responsible for useful characteristic displayed by host bacterium eg., antibiotic resistance. Figure 1.2: Bacterial Cell with Plasmid DNA and Chromosmal DNA Genetic Engineering & Applications (22BT52) 6 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering Figure 1.3: Antibiotic Resistance as a Selectable Marker for Plasmids Characteristics of Plasmids – a. It should be small (40 kb), supercoiled, covalently closed circular (CCC) DNA and exist as an extrachromosomal element. b. It should carry a multiple cloning site or polycloning region for insertion of the gene of interest. c. It should possess atleast two selectable markers one of which may be an antibiotic resistance gene. d. It should be present in large numbers per cell. e. It must have an origin of replication. f. It may or may not contain a promoter to express the gene of interest. Classification of Plasmids – A) Based upon the number of copies per cell, plasmids are classified into two types: 1. Stringent plasmids These plasmids exist in small numbers, i.e. 100 copies/cell. Relaxed plasmid is not under the control of bacterial genome for replication and segregation. Generally, relaxed plasmids are of low molecular weight and most of them are of the non-conjugative type. The most widely used method to find the copy number of the plasmid is to estimate the amount of enzyme encoded by genes present in the plasmid. For example, J3-lactamase activity can be measured if the plasmid specifies ampicillin resistance. Sometimes plasmids are also classified into compatible groups, based upon plasmid incompatibility. Plasmid incompatibility is the inability of two different plasmids to co exists in the same cell in the absence of selection pressure. But this method is not widely used. B) Based on replication strategies plasmids are classified into: Non integrative plasmids: All plasmids have atleast 1 DNA sequence that act as Origin of Replication----- replicate independently Genetic Engineering & Applications (22BT52) 7 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering Integrative plasmids/Episomes: In these plasmids, the dNA integrates into the host DNA and replicates along with it. Non Integrative plasmid Plasmids Cell Division Episome Plasmid Chromosome carrying integrated palsmid Bacterial Chrom Cell Division REPLICATION STRATEGIES Figure: 1.4: Size : The size of the plasmids range from 1.0kb (smallest)-250kb (largest) However desired size for cloning is that a plasmid should be less than 10kb. Copy Number The number of molecules of individual plasmid found in single bacterial cell is the copy number. They range from 1to more than 50 It is desirable that a plasmid makes multiple copies and large recombinant DNA molecules are produced. Plasmid examples: TYPES OF PLASMID VECTORS: Based on main characteristic coded by plasmid genes plasmid vectors are of 3 types:- Genetic Engineering & Applications (22BT52) 8 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering ‘F’plasmids are responsible for conjugation. They are also called (Fertility)/conjugative/transmissible plasmids. These plasmids Carry tra(transfer) genes responsible for conjugation. Their presence in bacteria gives them maleness. ‘R’ plasmids/resistance: carry genes for resistance to antibiotics. Col Plasmids: carry genes coding for colcins- proteins that kill sensitive bacteria. Eg ColE1 of E.coli. Plasmids in a bacterial cell can occur singly or in multiple copies. Single copy plasmids multiply and segregate with bacterial chromosome. This is called stringent replication. The multiple copy plasmids undergo more than one replication for each replication of bacterial chromosome. This is called relaxed replication. Examples of Plasmids pBR 322, PUC series, pET, pRSET a. pBR 322: pBR322 is a reconstructed plasmid. pBR322 is one among the most popular and widely used plasmids. It was constructed by using both classical genetic techniques and recombinant DNA methodology. It is one of the most versatile plasmids with characteristics of an ideal plasmid. pBR 322 is 4363 bp in length with ColE1 as replication start and with two antibiotic resistance genes (ampillicin and tetracycline). pBR322 was the first artificial plasmid. Created in 1977, it was named eponymously after its Mexican creators, p standing for plasmid, and BR for Bolivar and Rodriguez. Structure of E.coli plasmid vector: pBR322 It has the following regions: 1. Origin of Replication: It is derived from plasmid pMBI. This plasmid is closely related to naturally occurring plasmid Col E1. 2. Gene conferring resistance to antibiotics Ampicillin (amp r) and tetracycline (tetr) : Gene amp r is derived from plasmidR1 and gene tetr from R6.5. Both are natural populations’ antibiotic resistance plasmids. These genes are used as markers. 3. Unique recognition sites for 30 restriction endonucleases: Some of these lie within markers amp r and tetr genes. The presence of restriction sites within markers amp r and tetr permits easy Genetic Engineering & Applications (22BT52) 9 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering selection of transformed cells having recombinant pBR322. Insertion of DNA fragment into the plasmid using restriction enzyme Pst1 or Pvu1 places the DNA insert (Foreign DNA) with the marker gene amp r. The DNA insert within the marker gene amp r makes it non functional. Bacterial cells with such recombinant pBR322 will be unable to grow in the presence of ampicillin but will grow successfully in tetracycline. Similarly when restriction enzyme Bam H1 or Sal 1 is used, the DNA insert within the marker gene tet r. This makes tetracycline gene non functional. Transformed bacterial cells possessing such recombinant pBR322 will be able to grow in the presence of ampicillin but will not grow in tetracycline. This process allows an easy selection of single bacterial cell having recombinant DNA. Nomenclature of pBR322: The name pBR is derived from i) p denotes plasmid ii) B is from the name of the, Boliver and iii) R from Rodriguez. These two scientists developed the plasmid. iv) The numerical 322 distinguishes this plasmid from other plasmids developed in the same laboratory latter on such as pBR325, pBR327, pBR328. Useful feature of pBR 322:  Small size (4.4kb) enables easy handling, purification and manipulation  Two selectable markers permit easy selection of recombinant DNA.  About 15 copies of them per cell which can be increased to 1000 to 3000 when protein synthesis is blocked.  It is a cloning vector. Screening of pBR322:  During screening of pBR322, the gene of interest is spliced into tetracycline gene cluster, and then the E.coli cells are transformed. Thus, three types of cells are obtained. They are as follows:  Cells that have not been transformed and so contain no plasmid molecules and will be ampicillin and tetracycline sensitive.  Cells that have been transformed with pBR322 but without the inserted DNA fragment or gene will be ampicillin and tetracycline resistance. These are transformed cells.  Cells that contain a recombinant DNA molecule, that is, the DNA fragment has been inserted into the pBR32 at tetracycline gene cluster. These cells will lose tetracycline resistance because the fragment has inserted in the middle of tetracycline resistance gene cluster. These are recombinants and will be ampicillin resistance but tetracycline sensitive.  The bacteria are spread on agar medium and colonies grown. By checking for growth on media with ampicillin, except untransformed cells, both transformants and recombinants will produce colonies. Plates are then grown on media with ampicillin and tetracycline. Colonies that do not grown on tetracycline medium are recombinants whereas both transformants and recombinants will grow ampicillin medium. By comparing the replica plates, recombinants can be picked up from ampicillin agar plates. Genetic Engineering & Applications (22BT52) 10 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering b. pUC plasmid These are plasmid derived vectors. pUC plasmids stand for plasmids developed at University of California. pUC plasmid was constructed by J. Messing and J.Vieira. This plasmid contains 40% DNA similar to pBR322 and has ampillicin resistance gene. It has a short stretch of DNA with a cluster of restriction enzyme recognition sites. This stretch is called as "multiple cloning site" (MCS). These plasmids are 2,700 bp long and possess:  Ampicillin resistance gene,  The origin of replication derived from pBR322 and  The lac Z gene derived from E. coli. The lac Z gene comprises of short sequence for multiple unique cloning sites i.e. EcoRI, SacI, XmaI, Sma, BamHI, pstI, HindIII etc. and origin of replication. The gene lacZ is derived from lac operon of E.coli that codes for β-galactosidase enzyme. Which cleaves lactose into glucose and galactose A recombinant pUC molecule is constructed by inserting new DNA (gene) into one of the restriction sites that are clustered near the start of the lacZ site. The main advantages of pUC in genetic engineering are easy selection during screening process. Screening of pUC19: Selection of recombinant clones is based on the appearance of visible colored colonies. This visual screening involves uses the action of gene expressed products on a chromogenic substance to differentiate recombinant and non- recombinant clones. When bacteria are plated on the medium with X-gal and IPTG, the 13galactosidase enzyme will break X-gal (colorless) to release galactose and X- (indigo dye), which stains the bacterial colony blue. On the other hand if we have interrupted the plasmids by placing a gene of interest is the multiple Genetic Engineering & Applications (22BT52) 11 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering cloning sites, the gene becomes inactivated. Hence the bacterium fails to be stained and remains white. Thus, it is a simple matter to pick the colonies with inserts as they are the white ones and bacteria without insert are blue. Notice that in relation to cloning an insert in pBR 322, it is easier to clone in pUC vectors as we have to just look for a clone which is growing on the medium with white in the presence of X-gal. The cloning vectors belonging to pUC family arc available in pairs with reversed orders of restriction sites relative to lac Z promoter. pUC8 and PUC9 make one such pair. Other similar pairs include pUC12 and pUCl3 or pUC18 and pUC19. B. COSMIDS A cosmid, first described by Collins and Hohn in 1978, is a type of hybrid plasmid (often used as a cloning vector) that contains cos sequences, DNA sequences originally from the Lambda phage. li can be used to build genomic libraries. Artificially constructed cloning vector containing the cos gene/cos site of phage lambda. Cosmids can be packaged in lambda phage particles for infection into E. coli; this permits cloning of larger DNA fragments (up to 45 kb) than can be introduced into bacterial hosts in plasmid vectors. Cosmids and cosmid recombinants replicate as plasmids. Likely to be less stable than plasmids because of large insert and high copy number. Structure: A typical cosmid vector has following regions:  Origin of replication  Unique restriction site for cleavage and insertion of foreign DNA  A selectable marker from plasmid, coding for antibiotic resistance  Cos site/site from lambda phage has 12 bases. It enables recombinant DNA to be packed in the lambda particle in vitro due to circularization and ligation. Characteristics of cosmid vectors: The typical features of a cosmid vector are:  Cosmids have a length of about 5kb. They can be used to clone DNA inserts of upto 40- 45kb.  These can be packaged into λ-particles which infect host cells.  Packaged plasmids infect host cells like λ-phage particles, but mutiply and propagate like plasmids.  Selection for recombinant DNA is based on the procedure used for the selection of plasmids.  Cosmid vectors are amplified and maintained in the same way as plasmids. Use of cosmids: Cosmids are used for construction of genomic libraries of eukaryotes. Egs.,: PjBB, pWE, sCos Genetic Engineering & Applications (22BT52) 12 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering Cosmid structure ori Cos site Λ DNA Cloning using a cosmid: A Cloning experiment with a cosmid is carried out as follows: The Cosmid is opened at its unique restriction site and a new DNA fragment inserted. These fragments are produced usually by partial digestion with restriction endonuclease, as total digestion almost invariably results in fragments that are too small to be cloned with a cosmid. Ligation is carried out so that the concatamers are formed. Providing the inserted DNA is the right size, in vitro packaging will cleave the cos sites and place the recombinant cosmids in mature phage particles. These λ phages are the used to infect an E.coli culture, though of course plaques are not formed. Instead, infected cells are plated onto a selective medium and antibiotic resistant colonies are grown. All colonies are recombinants as non- recombinant linear cosmids are too small to be packaged into λ heads. C. PHAGEMIDS A phagemid or phasmid is a type of cloning vector developed as a hybrid of the filamentous phage M13 and plasmids to produce a vector that can grow as a plasmid, and also be packaged as single stranded DNA in viral particles. Phagemids contain an origin of replication (ori) for Genetic Engineering & Applications (22BT52) 13 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering double stranded replication, as well as an f1 ori to enable single stranded replication and packaging into phage particles. Structure: Origin of replication (ori) from phage f1 or M13 for Single Strand replication Many contain OR from phage hence called phagemids Multiple cloning site (MCS) within lacZ gene A portion of lacZ gene driven by lacZ promoter Phage T7 and T3 promoter Sequence that flank MCS Sequence Col E1 origin of replication (II) Ampr gene for antibiotic resistance DNA insert is integrated in vitro into double stranded vector. The vector is then introduced into E.coli cells like any other plasmid vector. It can be used for cloning DNA insert upto 10kb only. The recombinant DNA propagates inside E.coli as a plasmid using col EI origin of replication of plasmid and several copies of recombinant DNA are obtained. The phagemid vector can also be used as an expression vector because the DNA insert is transcribed along with lac Z gene. A fusion protein is formed which has lac Z gene product along with insert DNA product. 2. Construction of Vectors a. Blue script: pBluescript vector is an example of a combination between plasmids and phages, thereby gaining it a place within the catergory of vectors known as phagemids. Phagemids represent a hybrid type of class of vectors that serve to produce single-stranded DNA. pBluescript is a popular variety of phagemid commercially available to molecular biology labs for cloning purposes. Through the following descriptions of some key parts of pBluescript, characteristics of plasmids and phages (as discussed above) should be evident. Some useful features of pBluescript: Like pUC vectors, which pBluescript is derived from, there is a multiple cloning site inserted into the LacZ' gene, so clones with inserts can be distinguished as blue versus white cells by staining using X-gal. pBluescript also contains the origin of replication of the single-stranded phage f1, which is related to M13 phage vectors. This translates into that a cell harboring a recombinant phagemid, if infected by f1 helper phage that supplies the single-stranded phage DNA replication components; it will produce and package single- stranded phagemid DNA. A final feature of this class of phagemid vectors stems from the fact that it is flanked by two different phage RNA polymerase promoters. More specifically, pBS has a T3 promoter on one end side and a T7 promoter on the other terminal end. This is key because it enables one to isolate the double-stranded phagemid DNA and transcribe it in vitro with either of these two phage polymerases to produce pure RNA transcripts to coincide with either of the two different strands. Key Points:  2961 basepair plasmid  Derived By Replacing pUC19 polylinker of pBS (+/-) with synthetic polylinker  SK designation indicates that the polylinker is oriented such that the lacZ transcription proceeds from Sac I to Kpn I.  21 unique restriction sites in the multiple cloning region  Blue/White color selection  In vitro RNA transcription with T3 or T7 RNA polymerase Genetic Engineering & Applications (22BT52) 14 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering  Double and Single-stranded sequencing Figure: A general picture of pBluescript. C. Artificial Chromosme vectors What is an artificial chromosome? A type of cloning vector that has some features of true chromosomes and is used to clone relatively large fragments of DNA Artificial Chromosome vectors are Linear/circular shuttle vectors that occur as 1-2 copies per cell. They allow cloning of sequences that are several hundred kilobase pairs (up to 1000kb or more). These are of following types: Bacterial Artificial chromosome (BAC) Yeast Artificial chromosome (YAC) P1-derived Artificial chromosome (PAC) Mammalian Artificial chromosome (MAC) Human Artificial chromosome (HAC) Of these YAC is used for cloning in yeast cells, BAC & PAC in bacteria, MAC & HAC are used for mammalian and human cells. 1. Bacterial Artificial Chromosome (BAC) Vector: BAC vectors are shuttle plasmid vectors created for cloning large sized foreign DNA. They have origin of replication (oriS) from bacterium E.coli F-factor. This maintains a strict control on the copy number of vectors to one or two per cell. The low copy number of BAC avoids any possibility of recombination between different DNA inserts of multiple vectors. Structure: The first BAC vector was pBAC108L. Other BAC vectors are PBeloBAC11, PBACe3.6 etc. The vector PBeloBAC11 has 7.4kb and allows selection of recombinant clones by lacZ ᾱ complementation. It has the following modules: oriS origin of replication from E.coli F1 plasmid repE encodes replication protein that binds to oriS to initiate replication CMr chloramphenicol resistance as a marker cosN λ phage cos site lacZ, β-galactosidase gene as a restriction site T7 from bacteriophage for T7 RNA polymerase driven promoter Genetic Engineering & Applications (22BT52) 15 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering SP6, bacteriophage SP6 RNA polymerase driven promoter parA, parB, & parC for division of F plasmid during cell division IoxP site on phage P1 genome for recombination Importance of BAC Vectors: BAC vectors have emerged as more popular cloning vectors because of the following features: BACs vectors can clone DNA inserts of upto 300 kb They are stable and user friendly They do not suffer from chimerism caused by variation in cloned DNA recombination Low copy number of BAC vectors/host cell maintains DNA inserts in their original form. It also avoids counter section of cloned genes. BAC vectors are used extensively in analysis of genome Host for BAC vectors is a mutant strain of E.Coli in which normal restriction & modification sites are missing. 2. Yeast Artificial Chromosomes YACs are synthetic double stranded linear constructs that behave like a yeast chromosome. YACs contain all the elements necessary for replication as independent chromosomes in yeast. A typical YAC (eg., pYAC3) contains following functional modules from yeast chromosome: An autonomous replication sequence (ARS): ARS1, chromosome III ARS, ARSH4 A centromere: CEN4, CEN6: centromeres consist of three centromere determining elements, CDE I, CDE II, and CDE III. A telomeric sequence: Telomeric sequences from yeast chromosome at the two ends against exonuclease action, consisting of 20-70 tandem repeats of 6 bases, 5’…..CCCCAA. Selectable Markers: Typically the chromosome also contains a selection marker such as TRP1, Lys2 or Ura3. Sequences from E. coli plasmid for selection and propagation of E. coli. SUP4, a selectable marker where DNA insert can be integrated. The First YAC vector developed was pYAC3. It is essentially a pBR322 plasmid vector into which the above mentioned Yeast sequences have been integrated. It propagates in E.coli in a circular form. The DNA insert is integrated. It propagates in E.coli within SUP4 region to generate linear YAC vector. The recombinant YAC introduced into TRPI - & URA3- yeast cells by protoplast transformation. The recombinant clones are identified by insertional inactivation of SUP4 detected by a simple color test. YACs can accommodate upto 2000kb of cloned DNA inserted into multiple cloning sites. Therefore, YACs are used for cloning very large (100-1,400kb) DNA segments. Applications: To study function and modes of expression of genes. YACS are propagated in mammalian cells, enabling functional analysis to be carried out in the organism in which the gene normally resides. Production of gene libraries DNA sequencing and mapping programs. Eg Human genome project Disadvantages:  Cloning efficiency is very low about 1000 clones per µg DNA. Hence cannot be used for complete generation of genomic libraries.  It is not possible to recover large amount of pure insert DNA from individual clones. 3. Mammalian Artificial Chromosomes Genetic Engineering & Applications (22BT52) 16 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering Mammalian artificial chromosomes (MACs) are conceptually similar to YACs, but instead of yeast sequences they contain mammalian or human ones. In this case the telomeric sequences are multimers (multiple copies) of the sequence TTAGGG, and the commonly used centromeric sequence is composed of another repeated DNA sequence found at the natural centromeres of human chromosomes and called alphoid DNA. Because the alphoid DNA is needed in units of many kilobases, these MAC DNAs are grown as YACs or, more recently, as BACs. When added to suitable cell lines, these MAC DNAs form chromosomes that mimic those in the cell, with accurate segregation and the normal complement of proteins at telomeres and centromeres. Their primary use is not in genome mapping but as vectors for delivery of large fragments of DNA to mammalian cells and to whole animals for expression of large genes or sets of genes. They are still in development, and although gene expression has been demonstrated they have not been used in a practical application. 4. P1-derived artificial chromosome The P1-derived artificial chromosomes (PACs) are DNA constructs that are derived from the DNA of P1 bacteriophage. They can carry large amounts (about 100-300 kilobases) of other sequences for a variety of bioengineering purposes. It is one type of vector used to clone DNA fragments (100- to 300-kb insert size; average, 150 kb) in Escherichia coli cells based on bacteriophage (a virus) P1 genome. 5. Human artificial chromosome A human artificial chromosome (HAC) is a microchromosome that can act as a new chromosome in a population of human cells. That is, instead of 46 chromosomes, the cell could have 47 with the 47th being very small, roughly 6-10 megabases in size, and able to carry new genes introduced by human researchers. Yeast artificial chromosomes and bacterial artificial chromosomes were created before human artificial chromosomes, which first appeared in 1997. They are useful in expression studies as gene transfer vectors and are a tool for elucidating human chromosome function. Grown in HT1080 (HT1080 is a human fibrosarcoma cell line) cells, they are mitotically and cytogenetically stable for up to six months. John J. Harrington, Gil Van Bokkelen, Robert W. Mays, Karen Gustashaw & Huntington F. Willard of Case Western Reserve University School of Medicine published the first report of human artificial chromosomes in 1997. They were first synthesized by combining portions of alpha satellite DNA with telomeric DNA and genomic DNA into linear microchromosomes. Plant Vectors Like bacteriophages, even plant viruses can transfer recombinant genes into plants. Plant viruses are attractive as vectors for several reasons.  Plant viruses adsorb to and infect cells of intact plants.  Relatively large amounts of virus can be produced from infected plants, leading to the prospect of large amount of foreign protein being expressed from recombinant viruses.  Some virus infections are systematic. They are spread throughout the whole plant. In some cases intact viruses are transported through the vascular system of plant. Two groups of plant viruses, cauliflower mosaic viruses (CaMV) and Gemini viruses, both DNA viruses. Genetic Engineering & Applications (22BT52) 17 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering 1. Cauliflower Mosiac Virus (CaMV) :  CaMV is a circular, double stranded DNA genome of about 8 kb.  It causes systemic infection in small groups of plant species. It contains three discontinuous duplex DNA, (two in one strand, one in the other). These are regions of sequence overlap.  DNA itself is infected and can commence infection by inoculating on the surface of the host plant.  CaMV genome contains eight closely packed reading frames. There are only two small intragenic regions which can be discarded and replaced by the insert. If any other region is removed or when a large sized insert is used, no infection takes place.  Thus, only a small size of insert of few hundred bp can be used as insert. This requirement of a small sized insert imposes limitations on the use of this vector for gene transfer. Even the use of helper virus coding for the lost genes when a large insert is placed will not restore their functions. The reasons are not clear. 2. T-DNA Plasmid :  Plants do not have plasmids indigenous to them as yeasts do. Hence, bacterial plasmids, which can transfer DNA to plants, were developed.  The best developed plasmid based vector to transfer DNA into the plant is Ti plasmid.  Ti plasmids occur naturally in gram negative bacteria called Agrobacterium tumefaciens.  Ti-plasmid is 2.5 kb in size with unique regions called A, B, C and D. Regions 13 and C are involved in plasmid replication. D region is involved in the transfer of DNA from the plasmid into plant. This region of D is 40 kb in length and is referred to as the virulence region.  vir genes always transfer a fixed region from the plasmid to the plant cell. Such region is called as T-DNA or transfer DNA. T-DNA is 13-25 kb in length and codes for tumor inducing proteins.  T - DNA integrates into host genome at random sites without any specificity. These cells are transformed and produce a tumor called a "crown gall".  T-DNA codes for 3 proteins, 2 of them are responsible for the synthesis of auxin and cytokinin. The other protein directs the synthesis of unusual amino acids or sugar derivatives called as opines. Opines are not synthesized by untransformed cells.  Ti-plasmids are classified into two types based upon whether they produce octopine or nepaline.  Ti-plasmid thus provides bacteria with two important resources a source of metabolite and the means to use that metabolite as a source of energy.  Ti-plasmids are remarkable because they stand as examples for the insertion of prokaryotic gene into a eukaryotic genome. The plasmid looks like a natural chimera as it contains two sets of genes, one active in bacteria and the other in plant.  The genes in the T - DNA segment are associated with transcription control signals that operate in plants while those in the remainder of the plasmid are under the control of bacterial promoters.  T - DNA transfer is remarkable from the point of view of inter-kingdom gene transfer. Although T - DNA is transferred, it has no role in the transfer mechanism. T - DNA region in both Ti and Ri plasmids are flanked by almost perfect 25 base pair direct repeat sequences.  Especially the right hand 25' bp sequence is compulsorily required for T - DNA transfer as they function in a ds-acting manner.  Any DNA sequence could be transferred to plant cells as long as it is flanked by the 25 bp repeat sequence in correct order by vir genes. Genetic Engineering & Applications (22BT52) 18 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering Viral Vectors Viral vectors are one of the major vehicles used by scientists in gene therapy to get their sequences expressed in the proper host. There are a myriad of possible viral vectors. This is an area of continual new development in gene therapy. Key Properties: Viral vectors are tailored to their specific applications but generally share a few key properties. Safety: Although viral vectors are occasionally created from pathogenic viruses, they are modified in such a way as to minimize the risk of handling them. This usually involves the deletion of a part of the viral genome critical for viral replication. Such a virus can efficiently infect cells but, once the infection has taken place, requires a helper virus to provide the missing proteins for production of new virions. Low toxicity: The viral vector should have a minimal effect on the physiology of the cell it infects. Stability: Some viruses are genetically unstable and can rapidly rearrange their genomes. This is detrimental to predictability and reproducibility of the work conducted using a viral vector and is avoided in their design. Cell type specificity: Most viral vectors are engineered to infect as wide a range of cell types as possible. However, sometimes the opposite is preferred. The viral receptor can be modified to target the virus to a specific kind of cell. Types of Viral Vectors i. Retrovirus: recombinant retroviruses ii. Lentivirus: The viral genome in the form of RNA is reverse-transcribed when the virus enters the cell to produce DNA, which is then inserted into the genome at a random position by the viral integrase enzyme. The vector, now called a provirus. iii. Adenoviruses: AV DNA does not integrate into the genome and is not replicated during cell division. Occasionally used in invitro Expts. iv. Adeno associated virus: (AAV) is a small virus which infects humans and some other primate species. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell. These features make AAV a very attractive candidate for creating viral vectors for gene therapy. v. Nanoengineered substances: Nonviral substances such as Ormosil have been used as DNA vectors and can deliver DNA loads to specifically targeted cells in living animals. (Ormosil stands for organically modified silica or silicate). i. Retroviruses Basics of the retrovirus virion and infection:  Retrovirus virions contain a protein capsid that is lipid encapsulated.  Virions range in diameter from 80 to 130 nm.  The viral genome is encased within the capsid along with the proteins integrase and reverse transcriptase.  The genome consists of two identical positive (sense) single-stranded RNA molecules ranging in size from 3.5 to 10 kilobases.  Following cellular entry, the reverse transcriptase synthesizes viral DNA using the viral RNA as its template.  The cellular machinery then synthesizes the complementary DNA which is then circularized and inserted into the host genome.  Following insertion, the viral genome is transcribed and viral replication is completed. Genetic Engineering & Applications (22BT52) 19 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering  The majority of retroviruses are oncogenic although the degree to which they cause tumors varies from class to class. The retroviral genome: The retroviral genome consists of little more than the genes essential for viral replication. The prototype and simplest genome to describe is that of the Moloney murine leukemia virus (MMLV) in contrast to the highly complex genomes of the HTLV and HIV retroviruses. Simplest genome The genome can be divided into three transcriptional units: gag, pol and env. gag region encodes genes which comprise the capsid proteins; pol region encodes the reverse transcriptase and integrase proteins; env region encodes the proteins needed for receptor recognition and envelope anchoring. long terminal repeat (LTR) regions found at each of the gene is an important feature of viral genome. LTR plays an important role in  initiating viral DNA synthesis &  its integration as well as regulating transcription of the viral genes MMLV genome: Structure and gene products of an integrated retroviral genome. LTR LTR ψ gag pol env Host cell DNA Transcription v v Primary transcript v v Translation Polypeptide A Proteolytic clevage Polypeptide B integrase Viral envelope protease proteins Reverse transcriptase Virus structural proteins Structure and gene products of an integrated retroviral Genome: Transcription of the retroviral DNA produces a primary transcript encompassing the gag, pol, and env genes. Translation produces a polyprotein, a single long polypeptide derived from the gag and pol genes, which is cleaved into six distinct proteins. Splicing of the primary transcript yields an mRNA derived largely from the env gene, which is also translated into a polyprotein, then cleaved to generate viral envelope proteins. Retroviral Vectors for Gene Transfer: MMLV MMLV vectors: These vectors have been used more than any other gene transfer vehicle. They are produced simply by replacing the viral genes required for replication with the desired genes to be transferred. Thus, the genome in retroviral vectors will contain an LTR at each end with the desired gene or genes in between. The most commonly used system for generating retroviral vectors consists of two parts, the retroviral DNA vector and the packaging cell line. Genetic Engineering & Applications (22BT52) 20 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering Retroviral DNA vectors are plasmid DNAs which contain two retroviral LTRs in the region internal to these LTRs for insertion of the desired gene. A portion of a retroviral plasmid DNA vector, LNSX, is shown below. The gene of interest is cloned into the multi-cloning site following the simian virus SV40 promoter (SV). As one can imagine, these plasmid DNAs can be manipulated to meet a variety of needs allowing for multiple applications and the design of very elegant vectors. Advantages and Disadvantages: Advantages Disadvantages High Transduction Efficiency Requires dividing cells for infectivity Insert Size up to 8kB Low Titers (106- 107) Integrates into host genome resulting in sustained Integration is random expression of vector In vivo delivery remains poor. Effective only Extremely well studied system when infecting helper cell lines Vector proteins not expressed in host ii. Lentiviruses  Lentiviruses are a subclass of Retroviruses. They have recently been adapted as gene delivery vehicles (vectors) due to their ability to integrate into the genome of non-dividing cells, which is the unique feature of Lentiviruses as other Retroviruses can infect only dividing cells.  The viral genome in the form of RNA is reverse-transcribed when the virus enters the cell to produce DNA, which is then inserted into the genome at a random position by the viral integrase enzyme.  The vector, now called a provirus, remains in the genome and is passed on to the progeny of the cell when it divides. The site of integration is unpredictable, which can pose a problem.  The provirus can disturb the function of cellular genes and lead to activation of oncogenes promoting the development of cancer, which raises concerns for possible applications of lentiviruses in gene therapy.  Lentivirus vectors have a lower tendency to integrate in places that potentially cause cancer than gamma-retroviral vectors. More specifically, one study found that lentiviral vectors did not cause either an increase in tumor incidence or an earlier onset of tumors in a mouse strain with a much higher incidence of tumors Moreover, clinical trials that utilized lentiviral vectors to deliver gene therapy for the treatment of HIV experienced no increase in mutagenic or oncologic events.  For safety reasons lentiviral vectors never carry the genes required for their replication. To produce a lentivirus, several plasmids are transfected into a so-called packaging cell line, commonly HEK 293.  One or more plasmids, generally referred to as packaging plasmids, encode the virion proteins, such as the capsid and the reverse transcriptase.  Another plasmid contains the genetic material to be delivered by the vector. It is transcribed to produce the single-stranded RNA viral genome and is marked by the presence of the ψ (psi) sequence. This sequence is used to package the genome into the virion. Genetic Engineering & Applications (22BT52) 21 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering iii. Adenoviral Vectors Introduction: Adenoviruses were discovered in 1953 as investigators hurriedly attempted to identify the causative agents of the common cold. There are currently 47 distinct serotypes and as many as 93 different particular varieties of adenovirus, all of which generally infect the ocular, respiratory or GI epithelium. In 1977, Frank Graham developed a cell line which enabled the first production of recombinant adenoviruses in a helper free environment. Since this time, adenoviral vectors have recevied much attention as gene transfer agents and currently offer a wide variety of gene therapy applications. Basic structure of the adenovirus virion: Adenoviral virions are icosahedral in shape, 70 to 90 nm in diameter, and are not enveloped. The viral capsid contains 252 protein components the majority of which are three proteins: fiber, penton based and hexon. Fiber and penton base proteins are important in receptor binding and cell internalization, whereas hexon comprises the majority of the viral capsid. The viral genome is large, consisting of a single double-stranded DNA molecule 36 to 38 kilobases in size. Viral DNA replication and transcription are complex, and viral replication and assembly occur only in the nucleus of infected cells. Mature virions are released by cellular disintegration. Adenoviral infection is a highly complex process. It is initiated by the virus binding to the cellular receptor. Internalization occurs via receptor-mediated endocytosis followed by release from the endosome. After endosomal release, the viral capsid undergoes disassembly as it journeys to the nuclear pore. Nuclear entry of the viral DNA is completed upon capsid dissociation, and the viral DNA does not integrate into the host genome but remains in an episomal state. ADENOVIRUS VECTORS Advantages Disadvantages High transduction efficiency Expression is transient (viral DNA does not integrate) Viral proteins can be expressed in host following vector Insert size up to 8kb administration High viral titer (1010-1013) In vivo delivery hampered by host immune response Infects both replicating and differentiated cells Genetic Engineering & Applications (22BT52) 22 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering FIGURE: Use of retroviral vectors in mammalian cell cloning. A typical retroviral genome (somewhat simplified here), engineered to carry a foreign gene (pink), is added to a host-cell tissue culture. The helper virus (not shown) lacks the packaging sequence, _ , so its RNA transcripts cannot be packaged into viral particles, but it provides the gag, pol, and env gene products needed to package the engineered retrovirus into functional viral particles. This enables the foreign gene in the recombinant retroviral genome to be introduced efficiently into the target cells iv. Vaccinia Vectors  Vaccinia virus has been utilized as a means for immunization against smallpox and a variety of other infectious agents.  Vaccinia virus is a member of the pox virus family. They are large brick shaped virions measuring 300-450 by 170-270 nm.  They are enveloped and contain an extremely large genome of 130-200 kilobases. This large genome enables large genes to be inserted into vaccinia-based vector.  Vaccinia vectors can infect a large variety of cell types, but a primary concern is their safety.  As with smallpox vaccination, the adverse reaction rate for administration of vaccinia vectors is 1 in 50,000 doses. Obviously, vaccinia vectors for gene therapy applications are limited to individuals not previously vaccinated against smallpox or are immune compromised.  Recombinant vector production is similar to the other vector systems that we had discussed requiring a viral vector DNA and a packaging cell line.  These vectors offer the potential to develop a large variety of gene therapy based vaccinations. Genetic Engineering & Applications (22BT52) 23 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering  Currently these applications are not as cost-effective as the more traditional vaccination protocols but this system might allow for the vaccination against diseases presently lacking a vaccine.  The system is also being examined in the treatment of HIV, although currently little attention is given to vaccinia-based systems in the field of gene therapy. v. Animal Vectors : a. Bovine Papilloma Virus Vector (BPV):  BPV causes warts (uncontrolled epithelial proliferation) in cattle. It is a member of a group of viruses which induces warts and papillomas in a range of mammals. BPV normally infects terminally differentiated squamous epithelial cells.  BPV has a capsid protein surrounding a circular double-stranded DNA of size 79 kb. 69% of this genome is important for viral function, whereas 31 % of the genome can be replaced by the insert.  The recombinant BPV is constructed by ligating the insert and BPV vector (69%) onto the pBR 322 plasmid, thus generating the shuttle vector containing plasmid ori site and virus replication sequences.  These shuttle vectors are multiplied in E.coli cells first and then they are transformed into mouse cell line.  It has been observed that if these sequences are removed prior to transfection, the vector exists at high copy number i.e., 200 copies per cell.  When transfected with pBR322 sequences, it exists at low copy number, i.e., less than 10 copies cell.  The major advantage of BPV is the generation of permanent cell line. As the infected cells are not killed, a stable plasmid number is found even when the insert is of large size.  The selection of transformants is very easy as they form a pile of cells on the transferred monolayer of cells called "Focus". The transformed cells are then selected by the presence of marker gene which is mostly the neomycin phospho transferase gene coding for resistance against G418. b. Baculovirus Vectors  Baculovirus Vectors - Baculovirus infects insects. This virus is rod shaped with a large double stranded genome. During normal infections, baculovirus produces nuclear inclusion bodies which consist of virus particles embedded in a protein matrix.  This protein matrix is encoded with the virus and is called polyhedrin and this polyhedrin accounts for 70% of total protein encoded by the virus as the transcription of the polyhedrin is driven by extremely active promoters.  Genetic manipulation of the viral DNA is not possible as it has a very large DNA with many restriction sites for a single enzyme. Hence, the gene of interest is cloned into the small recombination transfer vector and co transfected into insect cell lines along with the wild type of virus in the cell.  Homologous recombination takes place between the polyhedrin gene and our gene of interest.  Thus, our gene of interest will be transferred from the vector plasmid into the wild type of virus, polyhedrin gene will be transferred from the virus on the plasmid. This is something like displacement reaction.  This displacement of gene will not affect the replication of virus, as polyhedrin gene is not required for replication. Genetic Engineering & Applications (22BT52) 24 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering c. SV40 Virus  SV40 is an abbreviation for Simian vacuolating virus 40 or Simian virus 40, a polyomavirus that is found in both monkeys and humans. Like other polyomaviruses, SV40 is a DNA virus that has the potential to cause tumors, but most often persists as a latent infection.  The virus was first identified in 1960 in cultures of rhesus monkey kidney cells that were being used to produce polio vaccine. It was named for the effect it produced on infected green monkey cells, which developed an unusual number of vacuoles. Virology  SV40 consists of an unenveloped icosahedral virion with a closed circular dsDNA genome of 5kb.  The virion adheres to cell surface receptors of MHC class 1 by the virion glycoprotein VP1.  Penetration into the cell is through a caveolin vesicle.  Inside the cell nucleus the cellular RNA polymerase II acts to promote early gene expression. This results in an mRNA that is spliced into two segments. The small and large T antigens result from this.  The large T antigen has two functions: 5% will go to the plasma membrane of the cell and 95% will go back to the nucleus. Once in the nucleus the large T antigen binds three viral DNA sites, I, II, and III. Binding of sites I, and II auto regulates early RNA synthesis. Binding site II happens each cell cycle. Binding site I initiates DNA replication at the origin of replication.  Early transcription gives two spliced RNAs that are both 19s. Late transcription gives both a longer 16s, which synthesizes the major viral capsid protein VP1, and the smaller 19s, which gives Vp2, and Vp3 through leaky scanning. All of the proteins, besides the 5% of large T go back to the nucleus because assembly of the viral particle happens in the nucleus. Eventual release of the viral particle is cytolytic and results in cell death. Transcription The early promoter for SV40 contains three elements. The TATA box is the transcriptional start site. The 21 base-pair repeats contain six GC boxes and are the site that determines the direction of transcription. Also, the 72 base-pair repeats are transcriptional enhancers. When the SP1 protein interacts with the 21 bp repeats it binds either the first or the last three GC boxes. Binding of the first three initiates early expression and binding of the last three initiates late expression. The function of the 72 bp repeats is to enhance the amount of stable RNA and increase the rate of synthesis. This is done by binding (dimerization) with the AP1 (activator protein 1) to give a primary transcript that is 3' polyadenylated and 5' capped. Short notes pUC Plasmids –  pUC plasmids stand for plasmids developed at University of California.  pUC plasmid was constructed by J. Messing and J.Vieira.  This plasmid contains 40% DNA similar to pBR 322 and has ampicillin resistance gene.  It has a short stretch of DNA with a cluster of restriction enzyme recognition sites. This stretch is called as "multiple cloning site" (MCS).  Instead of tetracycline gene, pUC vectors have a lacZ gene coding for J3-galactosidase enzyme. This gene helps in identification of plasmid pUC with insert and without insert.  If these colonies are plated on a medium containing J3-galactosidase indicator, colonies with the pUC plasmid will change colour. Genetic Engineering & Applications (22BT52) 25 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering  When bacteria are plated on the medium with X-gal and IPTG, the 13-galactosidase enzyme will break X-gal (colourless) to release galactose and X- (indigo dye), which stains the bacterial colony blue.  On the other hand if the plasmids are interrupted by placing a gene of interest is the multiple cloning site, the gene becomes inactivated. Hence the bacteria fail to be stained and remain white.  Thus, it is a simple matter to pick the colonies with inserts as they are the white ones and bacteria without insert are blue. Notice that in relation to cloning an insert in pBR 322, it is easier to clone in pUC vectors as we have to just look for a clone which is growing on the medium with white in the presence of X-gal. Yeast Vectors - Yeast Episomal Plasmid - YEP –  One interesting yeast plasmid is called the 2u circle. The 2u circle is a 6.3 kb circular, extrachromosomal element found in the nucleus of most Saccharomyces cerevisiae strains. The 2u circle doesn't give cells that carry it any apparent selective advantage, but it is stably maintained at about 50 to 100 copies per haploid genome of the yeast cells. Like the host chromosomes, the 2u circle is coated with nucleosomes and replication is initiated by host replication enzymes once per cell cycle. The origin of bidirectional DNA replication is initiated at a specific site on the plasmid called an ARS sequence ("autonomous replication sequence").  These are naturally available plasmids of size 6.3 kb. This plasmid is also called as 2 μm plasmid.  YEP contains an origin of replication and three genes, REP3 (cis-acting elements) REP2 and REPI. 50-100 copies of plasmids exist per cell, which represent 2-4% of total yeast genome.  About 50% of YEP is essential for replication and maintenance and the remaining is dispensable. These plasmids have no markers and both the halves are useful. Hence Beggs in 1978 constructed a shuttle vector by using the essential portion of 2 μM plasmid and pBR 322.  The marker gene encodes the enzyme required for histidine biosynthesis. Hence the recombinant transformants were selected by growing the yeast mutant for histidine biosynthesis on a medium lacking histidine.  Yep13 also includes entire pBR322 sequence, and therefore replicate and can be selected for both in yeast and E coli. Genetic Engineering & Applications (22BT52) 26 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering ENZYMES IN GENETIC ENGINEERING I. Introduction: The ability to manipulate DNA in vitro (outside the cell) depends entirely on the availability of purified enzymes that can cleave, modify and join the DNA molecule in specific ways. At present, no purely chemical method can achieve the ability to manipulate the DNA in vitro in a predictable way. Only enzymes are able to carry out the function of manipulating the DNA. Each enzyme has a vital role to play in the process of genetic engineering. The range of DNA manipulative Enzymes: These enzymes can be grouped into five broad classes depending on the type of reaction that they catalyze: 1. Nucleases are enzymes that cut, shorten or degrade nucleic acid molecules. 2. Ligases join nucleic acid molecules together. 3. Polymerases make copies of molecules. 4. Modifying enzymes remove or add chemical groups. 5. Topoisomerases introduce or remove supercoils from covalently closed –circular DNA. 1. Nucleases: Genetic Engineering & Applications (22BT52) 27 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering Nuclease is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids. Nucleases are further classified into two types based upon the substrate on which they act. a. DNAases: Nucleases which act on or cut the DNA are classified as DNases, b. RNases: Nucleases which act on the RNA are called as RNases. a. RNases: Ribonuclease A is an endoribonuclease that cleaves single-stranded RNA at the 3' end of pyrimidine residues. It degrades the RNA into 3'-phosphorylated mononucleotides and oligonucleotides. Some of the major use of RNase A are:  Eliminating or reducing RNA contamination in preparations of plasmid DNA.  Mapping mutations in DNA or RNA by mismatch cleavage. RNase will cleave the RNA in RNA:DNA hybrids at sites of single base mismatches, and the cleavage products can be analyzed. b. DNases: Deoxyribonuclease I cleaves double-stranded or single stranded DNA. Cleavage preferentially occurs adjacent to pyrimidine (C or T) residues, and the enzyme is therefore an endonuclease.  Major products are 5'-phosphorylated di, tri and tetranucleotides.  In the presence of magnesium ions, DNase I hydrolyzes each strand of duplex DNA independently, generating random cleavages.  In the presence of manganese ions, the enzyme cleaves both strands of DNA at approximately the same site, producing blunt ends or fragments with 1-2 base overhangs.  DNase I does not cleave RNA, but crude preparations of the enzyme are contaminated with RNase A; RNase-free DNase I is readily available.  Some of the common applications of DNase I are:  Eliminating DNA (e.g. plasmid) from preparations of RNA.  Analyzing DNA-protein interactions via DNase footprinting.  Nicking DNA prior to radiolabeling by nick translation. DNases are further classified into two types based upon the position where they act. Fig: The reactions catalysed by the two different kinds of nuclease. (a) An exonuclease, which removes nucleotides from the end of a DNA molecule. (b) An endonuclease, which breaks internal phosphodiester bonds. i) Exonucleases: DNases that act on the ends or terminal regions of DNA are called as exonucleases. They remove one nucleotide at a time from the end of a nucleotide molecule. i.e., exonucleases release nucleotides (Nucleic acid + sugar + phosphate); Exonucleases require a DNA strand with atleast two 5’ and 3’ ends. They cannot act on DNA which is circular. The main distinction between different exonucleases lies in the number of strands that are degraded when a double-stranded molecule is attacked.  Enzyme Bal31 (purified from bacterium Alteromonas espejiana) removes nucleotides from both the strands of the double stranded DNA molecule. The greater the length of time that Genetic Engineering & Applications (22BT52) 28 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering Bal31 is allowed to act on a group of DNA molecules, shorter the resulting DNA fragments will be.  E coli exonuclease III degrade just one strand of a double stranded molecule, leaving single- stranded DNA as a product. The enzyme is available as a recombinant product. Its Mol.wt is 28KD. The reactions catalysed by different types of exonuclease. (a) Bal31, which removes nucleotides from both strands of a double-stranded molecule. (b) Exonuclease III, which removes nucleotides only from the 3’terminus (see p. for a description of the differences between the 3’ and 5’ termini of a polynucleotide). ii) Endonuclease: Endonucleases break internal phosphodiester bonds within a DNA molecule. They act at a non-specific region in the centre of the DNA. Endonucleases can act on circular DNA and do not require any free DNA ends (i.e., 5 or 3 end). Moreover they release short segments of DNA. Classification of Endonucleases:  SI endonucleases (from Aspergillus oryzae) will cleave only single strands.  Deoxyribonuclease I (DNase I), which is prepared from cow pancreas, cuts both single and double-stranded molecules. Dnase I from bovine pancreas, 37KD, free from RNase is used for preparation RNA free from DNA. It is glycoprotein.  Restriction endonucleases: There are special group of enzymes called restriction endonucleases cleave double stranded DNA only at a limited number of specific recognition sites. Genetic Engineering & Applications (22BT52) 29 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering Fig: The reactions catalysed by different types of endonuclease. (a) S1 nuclease, which cleaves only single-stranded DNA, including single-stranded nicks in mainly double-stranded molecules. (b) DNase I, which cleaves both single- and double-stranded DNA. (c) A restriction endonuclease, which cleaves double-stranded DNA, but only at a limited number of sites.  T5 -5’exonuclease: It removes nucleotides after nucleotide to generate 5’P-mononucleotides. This enzyme is extensively used for deletion analysis of promoters to find out which part of the DNA segment can act as the promoter. If allowed for longer period it can generate single stranded DNA, but this enzyme does not act on single stranded DNA or RNA. This enzyme can also remove Apurinic and Apyrimidinic sites by endonuclease activity.  Lambda 5’ Exonuclease: This has the ability remove nucleotides one after another from the 5’ end of the ds DNA either from the blunt end or 5’ over hangs. This also useful in generating single stranded DNA and also for deletional analysis of the promoter elements. RESTRICTION ENDONUCLEASES: Enzymes that can cut (hydrolyse) DNA duplex at specific sites. Current DNA technology is totally dependent on restriction enzymes. Until 1970 there were no convenient methods available for cutting DNA into discrete, manageable fragments. 1970 - The Beginning of the Revolution. Discovery of a restriction enzyme in the bacterium Haemophilus influenza. In 1970 the first restriction endonuclease enzyme HindII was isolated. For the subsequent discovery and characterization of numerous restriction endonucleases, in 1978 Daniel Nathans, Werner Arber, and Hamilton O. Smith awarded for Nobel Prize for Physiology or Medicine. Since then, restriction enzymes have been used as an essential tool in recombinant DNA technology.  DNases which act on specific positions or sequences on the DNA are called as restriction endonucleases.  The sequences which are recognized by the restriction endonucleases or restriction enzymes (RE) are called as recognition sequences or restriction sites. These sequences are palindromic sequences (i.e., if read from right to left, the sequence is same, e.g. MADAM).  Three different classes of restriction endonucleases are recognized, each distinguished by a slight different mode of action. Based on the composition, enzyme cofactor, the nature of target sequence and position of their DNA cleavage site relative to target sequence the restriction enzymes are classified. Type I and type III are complex and have limited role in genetic engineering. Type II restriction endonucleases are the cutting enzymes that are important in gene cloning.  Restriction enzymes are endonucleases Genetic Engineering & Applications (22BT52) 30 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering Bacterial enzymes Different bacterial strains express different restriction enzymes The names of restriction enzymes are derived from the name of the bacterial strain they are isolated from  Cut (hydrolyse) DNA into defined and REPRODUCIBLE fragments  Basic tools of gene cloning  Names of restriction endonucleases Titles of restriction enzymes are derived from the first letter of the genus + the first two letters of the species of organism from which they were isolated. EcoRI - from Escherichia coli BamHI - from Bacillus amyloliquefaciens HindIII - from Haemophilus influenzae PstI - from Providencia stuartii Sau3AI - from Staphylococcus aureus AvaI - from Anabaena variabilis  Restriction enzymes recognize a specific short nucleotide sequence  The phosphodiester bond is cleaved between specific bases, one on each DNA strand  Restriction enzymes do not discriminate between DNA from different organisms Most restriction enzymes will cut DNA which contains their recognition sequence, no matter the source of the DNA  Restriction endonucleases are a natural part of the bacterial defence system  Part of the restriction/modification system found in many bacteria  These enzymes RESTRICT the ability of foreign DNA (such as bacteriophage DNA) to infect/invade the host bacterial cell by cutting it up (degrading it)  The host DNA is MODIFIED by METHYLATION of the sequences these enzymes recognise o Methyl groups are added to C or A nucleotides in order to protect the bacterial host DNA from degradation by its own enzymes Fig 7-5b, Lodish et al (4th ed)  Hundreds of restriction enzymes have been isolated and characterized Genetic Engineering & Applications (22BT52) 31 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering  Enables DNA to be cut into discrete, manageable fragments  Type II enzymes are those used in the vast majority of molecular biology techniques  Many are now commercially available  Each restriction enzyme will recognize its own particular site  Some recognize very short sequences consisting of only 4 base pairs. These tend to cut DNA more frequently (generating smaller fragments) as the likelihood that any stretch of DNA sequence will contain these minimal recognition sites is high. Approximately 1 site per 256 bases ([1/4]4)  Some require longer recognition sequences (up to 8 bp). The longer the recognition sequence the less frequently these sites are likely to occur in any particular DNA sequence. Enzymes which cut DNA very infrequently are known as RARE cutters. An 8 bp recognition site will occur approximately 1 per 65,536 bases ([1/4]8)  The sites occur more randomly than predicted, so that digestion by any one enzyme will generate DNA fragments of different lengths  Some recognize more than one sequence  There are restriction enzymes which allow substitutions in one or more positions of their recognition sequences.  Most common substitutions o purines (A or G), designated R o pyrimidines (C or T), designated Y o any nucleotide, designated N  Restriction enzymes are a useful tool for analyzing Recombinant DNA After ligating a particular DNA sequence into a cloning vector, it is necessary to check that the correct fragment has been taken up. Sometimes it is also necessary to ensure that the foreign DNA sequence is in a certain orientation relative to sequences present in the cloning vector.  Checking the size of the insert  Checking the orientation of the insert  Determining pattern of restriction sites within insert DNA Types of restriction enzymes A. Type I:  Type I restriction enzymes were the first to be identified and are characteristic of two different strains (K-12 and B) of E. coli.  These enzymes cut at a site that differs, and is some distance (at least 1000 bp) away, from their recognition site.  The recognition site is asymmetrical and is composed of two portions—one containing 3–4 nucleotides, and another containing 4–5 nucleotides—separated by a spacer of about 6–8 nucleotides.  Several enzyme cofactors, including S-Adenosyl methionine (AdoMet), hydrolyzed adenosine triphosphate (ATP), and magnesium (Mg2+) ions, are required for their activity.  Type I restriction enzymes possess three subunits called HsdR, HsdM, and HsdS; HsdR is required for restriction; HsdM is necessary for adding methyl groups to host DNA (methyltransferase activity) and HsdS is important for specificity of cut site recognition in addition to its methyltransferase activity. B. Type III: Type III has intermediate properties between type I and type II. Break both DNA strands at a defined distance from a recognition site a. e.g. HgaI b.Require Mg2+ and ATP Genetic Engineering & Applications (22BT52) 32 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering c. They cut DNA about 20-30 base pairs after the recognition site. d.These enzymes contain more than one subunit and require AdoMet and ATP cofactors for their roles in DNA methylation and restriction, respectively C. Type II: Type II recognises a specific target sequence in DNA, and then break the DNA (both strands), within or close to, the recognition site.  They are composed of only one subunit; their recognition sites are usually undivided and palindromic and 4–8 nucleotides in length.  They recognize and cleave DNA at the same site, and they do not use ATP or AdoMet for their activity.  They usually require only Mg2+ as a cofactor.  These are the most commonly available and used restriction enzymes.  Many restriction endonucleases recognize hexanucleotide target sites, but cut at four, five or even eight nucleotide sequences.  Eg., Sau3A (Staphylococcus aureus strain 3A) recognizes GATC, and Alu) (Arthrobacter luteus) cuts at AGCT. EcoRI recognition site (sticky end) The exact nature of the cut produced by restriction endonuclease is of considerable importance in the design of a gene cloning experiment. Two types of ends are produced by cleavage of DNA with different restriction enzymes. They are: i) Blunt ends: Restriction endonucleases make a simple double strand cut in the middle of the recognition sequence resulting in a blunt or flush end. Eg., PvuII and AluI. ii) Sticky Ends or cohesive ends: Large number of restriction endonucleases cut DNA in a slightly different way. The two DNA strands are not cut at exactly the same position. Instead the cleavage is staggered, usually by two or four nucleotides, so that the resulting DNA fragments have short single-stranded overhangs at each end. These are called sticky or cohesive ends, as base pairing between them can stick the DNA molecule back together again. Restriction endonucleases with different recognition sequences may produce the same sticky ends. Eg., BamHI (recognition sequence GGATCC) and BglII (AGATCT) both produce GATC sticky ends. The same sticky end is produced by Sau3A, which recognizes only the tetranucleotide GATC. Fragments produced by cleavage with either of these enzymes can be joined to each other, as each fragment will carry a complimentary sticky end. The frequency of recognition sequences in a DNA molecule: The number of recognition sequences for a particular restriction endonuclease in a DNA molecule of known length can be calculated mathematically. A tetranucleotide sequence (eg., GATC) should occur every 44=256 nucleotides, and a hexanucleotide (GGATCC) once every 46=4096 nucleotides. However, only experimental analysis can provide the true picture Genetic Engineering & Applications (22BT52) 33 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering  Different restriction enzymes present in different bacteria can recognize different or same restriction sites. But they will cut at two different points within the restriction site. Such restriction enzymes are called as isoschizomers. Interestingly no two restriction enzymes from a single bacterium will cut at the same restriction site.  Isoschizomers are pairs of restriction enzymes specific to the same recognition sequence. For example, Sph I (CGTAC/G) and Bbu I (CGTAC/G) are isoschizomers of each other. The first enzyme to recognize and cut a given sequence is known as the prototype, all subsequent enzymes that recognize and cut that sequence are isoschizomers. Isoschizomers are isolated from different strains of bacteria and therefore may require different reaction conditions.  Neoschizomer: An enzyme that recognizes the same sequence but cuts it differently is a neoschizomer. Neoschizomers are a specific type (subset) of Isoschizomers. For example, Sma I (CCC/GGG) and Xma I (C/CCGGG) are neoschizomers of each other.  Isocaudomer: An enzyme that recognizes slightly different sequence, but produces the same ends is an isocaudomer.  In some cases, only one out of a pair of isoschizomers can recognize both the methylated as well as unmethylated forms of restriction sites. In contrast, the other restriction enzyme can recognize only the unmethylated form of the restriction site. This property of some isoschizomers allows identification of methylation state of the restriction site while isolating it from a bacterial strain. For example, the restriction enzymes HpaII & MspI are isoschizomers, as they both recognize the sequence 5'-CCGG-3' when it is unmethylated. But when the second C of the sequence is methylated, only MspI can recognize both the forms while HpaII cannot.  Star activity is a relaxation or alteration of the specificity of restriction enzyme mediated cleavage of DNA that can occur under reaction conditions that differ significantly from those optimum for the enzyme. The result is typically cleavage at non-canonical recognition site, or sometimes complete loss of specificity. Genetic Engineering & Applications (22BT52) 34 Dr N Rajeswari  Department of Biotechnology, Dayananda Sagar College of Engineering Differences which can lead to star activity include low ionic strength, high pH, and high (> 5% v/v) glycerol concentrations. The latter condition is of particular practical interest, since commercial restriction enzymes are usually supplied in a buffer containing a substantial amount of glycerol (50% v/v is typical), meaning insufficient dilution of the enzyme solution can cause star activity; this problem most often arises during double or multiple digests. Star activity can happen because of presence of Mg2+, for example, star activity in HindIII. Comparative properties of restriction enzymes: Property Type I RE Type II RE Type III RE Less common than Abundance Most common Rare Type II Cut both strands at a Cleavage of one Cut both strands at a non- specific, usually strand, only 24-26 bp Recognition site specific l