Dr. B. D. Ranjitha Kumari Professor and Head Department of Botany Bharathidasan University

1. Recombinant DNA Technology Dr. B. D. Ranjitha Kumari Professor and Head Department of Botany Bharathidasan University Tiruchirappalli – 620 024 Tamil Nadu, India

2. Restriction Endonucleases: • Recombinant DNA technology is Genetic Engineering which effects artificial modification of genetic constitution of a living cell by introducing of foreign DNA through experimental techniques. • Bacteria contains nucleases, that cleave the DNA and cleave the DNA backbone at specific sites on both strands of the duplex. These enzymes are called Type II Restriction Endonucleases or simply Restriction Enzymes (RE).

3. BIOLOGY OF THE RESTRICTION ENZYMES • The phenomenon of restriction-modification in bacteria is a small scale immune system for protection from infection by foreign DNA. • In higher organisms identification and inactivation of parasites is performed extracellularly. But bacteria can protect themselves only after foreign DNA has entered their cytoplasm. • For this protection, many bacteria specifically mark their own DNA by methylating bases on particular sequences with modifying enzymes. • DNA that is recognized as foreign by its lack of methyl groups on these same sequences is cleaved by the restriction enzymes and then degraded by exonucleases to nucleotides.

4. Fig. Methylation of an asymmetrical sequence necessitates recognition and methylation of two different sequences on the daughter strands on DNA replication. Imagine that the double standard DNA contains methyl groups on both strands of the DNA at a recognition sequence. DNA replication creates a new duplex, in which one of the strands in each of the daughter duplexes at first lacks the modification. This half methylated DNA must not be recognized as foreign DNA and cleaved, but instead must be recognized as self and methylated. So if the DNA is methylated on one of the two strands, the modification system methylates the other strand; if the DNA is methylaed on both strands, the enzymes do nothing.

5. Fig. Difference between Un-methylated and Methylated Sequence.

6. Why don’t Bacteria destroy their own DNA with their Restriction Enzymes? • DNA of bacteria is structured into rings called plasmids, so there are no free ends for the enzyme to work on and also the restriction enzymes were evolved to be protective to the specific bacteria which probably don't have the restriction sequence that the enzymes is specific for. • The bacterium protects its own DNA from nucleolytic attack by methylating the bases at susceptible sites, a chemical modification that blocks the action of the enzyme.

7. Restriction sites commonly are short palindromicsequences, that is, the restriction-site sequence is the same on each DNA strand when read in the 5 → 3 direction Recognition Sites have Symmetry (Palindromic) For Example: WOMEN UNDERSTAND MEN; FEW MEN UNDERSTAND WOMEN

8. Palindromes , of course can be of any size but most that are utilized as Restriction – Modification recognition sequence as four, five, six and rarely eight bases long. Fig. Palindromic DNA sequences.

9. Restriction Endonucleases : Named for bacterial genus, species, strain, and type Example: EcoR1 Genus: Escherichia Species: coli Strain:R Order discovered: 1

10. Many restriction enzymes make staggered cuts in the two DNA strands at their recognition site resulting a single-stranded “tail” at both ends. The tails at both ends are complementary. • At room temperature, these single-stranded regions, often called “sticky ends,” they can base-pair with those on other DNA fragments generated with the same restriction enzyme. • A few restriction enzymes, such as AluI and SmaI, cleave both DNA strands at the same point within the restriction site, generating fragments with “blunt” (flush) ends in whichall the nucleotides at the fragment ends are base-paired to nucleotides in the complementary strand.

11. Types of Cleavage Example: Alu I and Sma I Example: EcoRI

12. Restriction Enzymes

13. Isolation of DNA Fragments: Following cleavage of DNA by Restriction Enzymes, DNA fragments frequently must be isolated as the double standard DNA fragments of the same length have the same shape therefore migrate during electrophoresis at a rate nearly independent of there sequence Following Electrophoresis, bands formed by the different sized fragments may be located by autoradiography if the DNA had been radiolabelled before separation. Usually 32PO4 is a convenient label because phosphate is found in RNA and DNA and 32P emits particularly energetic electrons making them easily detectable.

14. Joining DNA fragments: In Vivo, the enzyme DNA ligase repairs nicks in the DNA backbone. The same activity may also utilized in vitro for the joining of DNA molecules. First, the molecule must be the correct substrates and they must possess 31 hydroxyl and 51 – phosphate groups. The method for generating the proper positioning has two variations: Sticky Ends Either to hybridize the fragments to gather via there sticky ends (OR) Blunt Ends Flush – ended fragments are to be joined.

15. The flush ends of DNA molecules that are generated by some restriction enzymes generate problems . One solution is to convert flush ended molecules to sticky ended molecules by enzyme terminal transferase. This enzyme adds nucleotide to the 31 end of the DNA. Fig. Joining of two DNA fragments by Poly – dA and Poly – dT tails Poly – dA tails can be put on one fragment and Poly – dT tails can be added to the other fragment.

16. Linkers can also be used to generate self complementary single standard molecules . • Linkers are short flush ended DNA molecules containing the recognition sequence of a RE that produces sticky ends. • The ligation of linkers to DNA fragments proceeds with reasonably high efficiency because high molar concentrations of the linkers may easily obtained Fig. Addition of linkers by ligation and their conversion to sticky ends by RE digestion • After the linkers have been joined to the DNA segment, the mixture is digested with RE which cuts the linkers and generate the sticky ends . • In this way a blunt ended DNA molecules is converted to a sticky – ended molecule that may easily be joined to other DNA molecules.

17. VECTORS: • Cloning Vectors • The molecular analysis of DNA has been made possible by the cloning of DNA. The two molecules that are required for cloning are the DNA to be cloned and a cloning vector. • Vectors must fulfill the two requirements – • Replication in the host cell • Selections of the cells having received the transforming DNA. BASIC TYPE OF VECTORS

18. PLASMID VECTORS • Most plasmids are small circles that contain the elements necessary for DNA replication, one or two drug resistance genes and a region of DNA into which foreign DNA may be inserted without damage to essential plasmid functions. • A useful element to have on plasmids is a DNA replication origin from a single standard phage. When such an origin is activated by phage infection, the cell synthesizes sizeable quantities of just one standard of the plasmid. Fig. A map of the plasmid PBR322 showing the ampicillin and tetracycline resistant genes, the origin and a few restriction enzyme cleavage sites.

19. In a typical cloning experiment, a plasmid is cut in a nonessential region with a restriction enzyme such as Eco RI, foreign Eco RI – cut DNA is added, and the single stranded ends are hybridized together and ligated. • Only a small fraction of the plasmids subjected to this treatment will contain inserted DNA. • One method for identifying candidates relies on insertional inactivation of drug resistance gene • For Example: Within the ampicillin resistance gene in PBR322 exists the only plasmid cleavage site of the restriction enzyme PstI. • Fortunately Pst1 cleavage generate sticky ends and DNA may readily be ligated into this site, whereupon it inactivates the ampicillin resistance gene. • Tetracycline resistance gene on the plasmid remains intact and can be used for the selection of the cells transformed with the recombinant plasmid.

20. Another way to screen for the insertion of foreign DNA utilizes the β – galactosidase gene. • Insertion of foreign DNA within the gene inactivates the enzyme, which can be detected by plating transformed cells on medium that selects for the presence of the plasmid and also contains substrates of β – galactosidase that produce colored dyes when hydrolyzed. • Cloning vectors are designed for the insertion of Foreign DNA into the short, N – terminal part of β – galactosidase .

21. A simple technique can greatly reduce recircularizaion of vector molecules that lack inserted foreign DNA. If the vector DNA is treated with a phosphatase enzyme after cutting with the restriction enzyme, then circularization impossible because the 51 _ PO4 ends required by DNA ligase are absent. Foreign DNA, however will carry 51 _ PO4 ends required by DNA ligase are absent. Foreign DNA, however will carry 51 _ PO4 ends, and therefore two of the four breaks flanking a fragment of foreign DNA can be ligated. This DNA is active in transformation because cells repair the nick remaining at each end of the inserted fragment.

23. Uses for Restriction Enzymes • RFLP analysis (Restriction Fragment Length Polymorphism) • DNA sequencing • DNA storage – libraries • Transformation • Large scale analysis – gene chips

24. Applications: • Cloning DNA fragment, generating libraries, essential step for genome mapping. • Positional Cloning : Discovering Disease Genes, Discovering Genes from Protein Sequence • Enzymes for Manipulating DNA : • DNA Polymerases • Kinases • Alkaline Phosphatase • Nucleases • Topoisomerases • Nucleases • Exonucleases- Remove nucleotides one at a time from a DNA molecule. • Endonucleases – Break Phospodiester bonds within a DNA molecule include Restriction Enzyme.

25. DNA Sequencing • It is to identify genes, to determine the sequences of promoters , other regulatory DNA elements that control expression, help to deduce the amino acid sequence of a gene or cDNA from the DNA sequence. • Manual DNA sequencing by the sanger dideoxy DNA method

26. Basic steps involved: • Isolation of the gene to be cloned. • Insertion of the gene into another piece of DNA called vector which will allow it to be taken by bacteria and replicated within them as the cells grow and divide. • Transfer of the recombinant vector into bacterial cells, either by transformation or by infection using viruses. • Selection of those cells which contain the desired recombinant vectors. • Growth of the bacteria, that can be continued indefinitely, to give as much cloned DNA as needed. • Expression of the gene to obtain the desired product.

28. Recombinant dna technology • Direct Gene Transfer Or Vector Less Mediated • Physical methods 1. Microinjection 2. Electroporation 3. Particle Bombarment • Chemical method 1. liposome 2.Calcium Phosphate method 3.PEG 4.DEAE- Dextran

29. Indirect Gene Transfer Or Vector Mediated: • Agrobacterium as gene transfer system • Viral mediated gene transfer

30. Electroporation • Basically involve the use of electrical impulses to reversibly permeabilize the cell membranes for the uptake of DNA. • Deliver of DNA into intact plant cells and protoplasts by forming the pores in the plasma membrane. Advantage: • Transformed cells are at the same physiological state after electroporation. • Efficiency improved by optimizing the electrical field strength and addition of spermidine.

31. Microinjection • Delivery of nucleic acids to protoplasts or intact cells via microinjection is a labour intensive procedure that requires special capillary needles, pumps,micromanipulators, inverted microscope and other equipment. • This method involves skill of the worker to insert needle into the cytoplasm or in the nucleus. • In order to microinject protoplasts or other plant cells, the cells need to be immobilized. • A maximum of 100-200 cells per hour can be microinjected by this method.

32. Particle bambardment: • This is latest technology to transfer DNA into intact tissues. Several devices are developed using different methods. • In this procedure micron size tungsten or gold particles are accelerated in a gun barrel to velocities sufficient for non-lethal penetration of cell walls and membranes. • An acceleration device used to propel particles (micro projectiles) carrying plasmid DNA is called by various names based on machine or technique used to accelerate the particles such as ‘particle gun technology’, ‘biolistic method’, ‘DNA bombardment’, ‘particle acceleration of DNA method’ and ‘electric discharge particle acceleration method’.

33. Indirect method

34. Indirect method

35. Introduction • The Ti plasmids exits as independent replicating circular DNA molecules within the Agro bacterium cells. • They are of approximately 200 kb in size. • They are variable in length in the range of 12 to 24 kb, which depends on the bacterial strain from which Ti plasmid comes. • The Nopaline strains of Ti plasmid have one T-DNA with length of 20kb while Octopine strains have 2 T-DNA regions referred to as TL and TR that are respectively 14 kb and 7 kb in length.

37. T-DNA region: • This region has the genes for biosynthesis of auxin (aux), cytokinin (cyt) and opine (ocs) and is flanked by left and right borders. • These 3 genes- aux, cyto nad ocs are referred to as oncogenes, as they are the determinants of the tumor phenotype;

38. T-DNA borders: • A set of 24 kb sequences present on either side ( right and left) of T-DNA are also transferred to the plant cells. • It is now clearly established that the right borders is more critical for T-DNA transfer and tumorigenesis. 2. Virulence region: • The genes responsible for the transfer of T-DNA into the host plant are located outside T-DNA and the region is referred to as vir or virulence region.

39. Vir region codes for proteins involved in T-DNA transfer. At least 9 vir gene operons have been identified. • Thee include vir A, vir G, vir B1, vir C1, vir D1, vir D2, vir D4 and vir E1 and E2. 3. Opine catabolism region: • This region codes for protein involved in the uptake and metabolisms of opines. Besides, the above three there is ori region that is responsible for the orogin of DNA replication which permits the Ti plasmid to be stably maintained in A.tumefaciens.

40. T-DNA Transfer and integration • The process of T-DNA transfer and its integration into host plant genome is as follows:- • Signal induction to Agrobacterium: • The wounded plant cells release certain chemicals-phenolic compounds and sugars which are recognized as signals by Agrobacterium. • The signals induced result in a sequence of biochemical events in Agrobacterium that ultimately helps in the transfer of T-DNA of plasmid.

41. 2. Attachment of Agrobacterium to plant cells : • The Agrobacterium attaches to plant cells through polysaccarides, particularly cellulose fibres produced by the bacterium. Several chromosomal virulence (chv) genes responsible for the attactment of bacterial cells to plant cells have been identified.

42. 3. Production Of Virulence Proteins: • As the signal induction occurs in the Agrobacterium cells attached to plants cells, a series of events take place that result in the production of virulence proteins. • Signal induction by phenolics stimulates vir A which in turn activates vir G. This induces expression of virulence genes of Ti plamsid to produce the corresponding virulence proteins( D1, D2, E2, B etc.)

43. 4. Production of T-DNA strand : • The right and left borders of T-DNA are recognized by vir D1/vir D2 proteins.these proteins are involved in the production of single stranded T-DNA, its protection and export to plant cells. • The ss T-DNA gets attached to vir D2.

44. 5. Transfer of T-DNA out of Agrobacterium: • The ss T-DNA- vir D2 complex in association with vir G is exported from the bacterial cell. Vir B products form the transport apparatus.

45. 6. Transfer of T-DNA into plant cells and integration : • The T-DNA-vir D2 complex crosses the palnt plasma membrane. tn the plant cells, T-DNA gets covered with vir E2. • This covering protects the T-DNA from degradation by nucleases. Vir D2 and vir E2 interact with a variety of plant proteins which influences T-DNA transport and integration. • The T-DNA-vir D2-vir E2-plant protein complex enters the nucleus through nuclear pore complex.

46. Within nucleus, the T-DNA gets integrated into the plant chromosome through process referred to illegitimate recombination. • This is different from the homologous recombination, as it does not depend on the sequence similarity.

47. 2-4 weeks shoots regenerate kanamycin

48. AGROBACTERIUM RHIZOGENES

49. A. rhizogenes, the causative agent of hairy rootsyndrome, is a common soil bacterium (Gram negative) capable of entering a plant through a wound and causing a proliferation of secondary roots. • The underlying mechanism of hairy root formation is the transfer of several bacterial genes to the plant genome.

50. The observed morphogeniceffects in the plants after infection have been attributed to the transfer of part of a large plasmid known as the Ri (root-inducing) plasmid. • The symptoms observed with A.rhizogenes are suggestive of auxin effects resulting from an increase in cellular auxin sensitivity rather than auxin production.