MOLECULAR BIOLOGY I & II

1. MOLECULAR BIOLOGY I & II BTG 303 & BIO 305

2. RECOMBINANT DNA TECHNOLOGY • Joining together of DNA molecules from two different species that are inserted into a host organism to produce new genetic combinations (i.e recombinant DNA) that are of value to science, medicine, agriculture and industry

3. RESTRICTION ENDONUCLEASES AND OTHER ENZYMES USED IN GENETIC ENGINEERING

4. RESTRICTION ENDONUCLEASES • Also called restriction enzymes or molecular scissors • They are enzymes that cut DNA at or near specific recognition nucleotide sequences known as restriction sites • They are found in bacteria and archaea • A bacterium uses a restriction enzyme to defend against bacterial viruses called bacteriophages or phages.

5. When a phage infects a bacterium, it inserts its DNA into the bacterial cell so that it might be replicated. Restriction enzyme prevents replication of the phage DNA by cutting it into many pieces • The bacterial DNA is prevented from the action of the restriction enzyme by another set of enzymes known as DNA methyltransferases or methylases • DNA methylaseis synthesized by the bacteria. It adds methyl to the DNA sequence of the bacteria for protection against restriction enzyme • The combination of restriction endonuclease and methylase is called RESTRICTION-MODIFICATION SYSTEM

6. Restriction Enzymes Enzymes AluI BamHI EcoRI HindIII KpnI NotI Sau3A Sources Arthrobacterluteus Bacillus amyloliquefaciens Escherichia coli Haemophilusinfluenzae Klebsiellapneumoniae Nocardiaotitidis Staphylococcus aureus

7. Nomenclature • Each restriction endonuclease is named after the bacterium from which it was isolated using a naming system based on bacterial genus, species and strain • The first letter of the generic name and the first two letters of the specific name of the organism is used to form a three letters abbreviation. For example, Escherichia coli- Eco and Haemophilus influenza- Hin • The strain is identified by the next letter. For example, EcoRi.e from strain Ry13

8. The order of identification of the enzyme in a bacterium is designated with a Roman letter. For example, EcoRI ie first endonuclease to be discovered in Escherichia coli • EcoRI is from Escherichia (E) coli (co), strain Ry13 and first endonuclease (I) to be discovered. • HindIII is from Haemophilus (H) influenzae (in), strain Rd (d) and the third endonucleases (III) to be discovered.

9. Recognition Sequences • All restriction endonucleases require specific nucleotide sequences to carry out cleveage. These sequences are known as recognition sequences  • Characteristics of recognition sequences: 1. Length of recognition sequences varies. For example, EcoRI recognizes sequence of six base pair in length while NotI recognizes a sequence of eight base pair in length 

11. 2. Different restriction enzymes have the same recognition sequence. These are better known as isoschizomers. For example, SphI and BbuI restriction enzymes has the same recognition sequence. 

13. 3. Recognition sequence of one enzyme contains recognition sequence for the other. For example, the restriction enzyme BamHI also contains the recognition sequence for another enzyme Sau3AI.

15. 4. Recognition sequences are palindromic, meaning the base sequence reads the same backwards and forwards. There are two types of palindromic sequences that can be possible in DNA:

16. The mirror-like palindrome is a sequence that reads the same forward and backwards on a single strand of DNA strand, as in GTAATG. The inverted repeat palindrome is also a sequence that reads the same forward and backwards, but the forward and backward sequences are found in complementary DNA strands (i.e., of double-stranded DNA), as in GTATAC (GTATAC being complementary to CATATG). Inverted repeat palindromes are more common and have greater biological importance than mirror-like palindrome

17. TYPES • Naturally occurring restriction endonucleases are categorized into four groups (Types I, II III, and IV) based on their composition and enzyme cofactor requirements, the nature of their target sequence, and the position of their DNA cleavage site relative to the target sequence.

18. Type I • Type I restriction enzymes were the first to be identified and were first identified in two different strains (K-12 and B) of E. coli. • These enzymes cut at a site that differs, and is a random distance (at least 1000 bp) away, from their recognition site.

19. Type II • They recognize and cleave DNA at the same site • They are the most commonly available and used restriction enzymes • Some examples of the type II restriction endonucleases include BamHI, EcoRI, EcoRV, and Haelll

20. Type III • They cut DNA about 20-30 base pairs after the recognition site.

21. Type IV • Type IV enzymes recognize modified, typically methylated DNA

22. Cleavage Patterns The restriction enzymes generate three different types of ends after cleavage within the recognition sequences.   • 1. Sticky ends: The restriction enzymes cut asymmetrically within the recognition sequences leaving single -stranded overhangs and are also called as cohesive ends. There are two types of sticky ends. • a) 5’ overhangs: The restriction enzymes cut asymmetrically within the recognition sequence in such a way that it leaves an overhanging short single stranded 5’ end. For example, 

24. b) 3’ overhangs: The restriction enzymes cut asymmetrically within the recognition sequence in such a way that it leaves an overhanging single-stranded 3’ ends. For example, 

26. But, the limitation of the sticky end is that they only stick to fragments which are compatible. For example, two EcoRI fragments can join together but EcoRI cannot join with any other fragments produced by other restriction enzyme. • 2. Blunt ends: The restriction enzymes cleaves exactly on the same site on both the strands without leaving any overhangs. For example, 

28. The DNA fragments with sticky ends are particularly useful for recombinant DNA experiments as the single-stranded sticky ends can easily pair with any other DNA fragment having complementary sticky ends. • A selected list of enzymes, recognition sequences and their products having either sticky or blunt ends formed are given below 

29. List of Restriction Enzymes, Recognition Sequences and its Products. Enzymes          Recognition sequence 1. Alu1                        5' AGCT                                3' TCGA  2. BamH1                   5' GGATTC                                    3' CCTAAG 3. EcoRI                     5' GAATTC                                    3' CTTAAG 4. HindIII                    5' AAGCTT                                    3' TTCGAA Cuts 5'---AG                      CT--- 3' 3'---TC                      GA---5'     5'---G                 GATTC---3' 3'---CCTAA     G 5'---G                 AATTC---3' 3'---CTTAA                 G---5' 5'---A                 AGCTT---3' 3'---TTCGA                A---5'

30. 5. KpnI5' GGTACC 3' CCATGG 6. NotI5'CGGCCGC 3'GCCGGCG 7. PstI5' CTGCAG                                    3' GACGTC 8. Sau3A                    5' GATC                                    3' CTAG 5'---GGTAC                C---3' 3'---C                CATGG---5' 5'---GC         GGCCGC---3' 3'---CGGGGG         CG---5' 5'---CTGCA               G---3' 3'---G               ACGTC---5' 5'-------               GATC---3' 3'----CTAG                 -----5'

31. DNA LIGASE

32. This enzyme repairs broken DNA by joining two nucleotides in a DNA strand. It is commonly used in genetic engineering to do the reverse of a restriction enzyme, i.e. to join together complementary restriction fragments. • The sticky ends allow two complementary restriction fragments to anneal, but only by weak hydrogen bonds, which can quite easily be broken by gentle heating. The backbone is still incomplete.

33. DNA ligase completes the DNA backbone by forming covalent bonds. Restriction enzymes and DNA ligase can therefore be used together to join lengths of DNA from different sources.

34. CLONING VEHICLES (TYPES AND CONSTRUCTION)

35. CLONING • Production of multiple copies of a specific DNA molecule • Also used to describe the production of genetically identical cells or even organisms

36. CLONING VEHICLE • Also known as cloning vector • A length of DNA that carries the gene to be cloned into a host cell • A cloning vehicle is needed because a length of DNA containing a gene on its own can not actually do anything inside a host cell. Since it is not part of the cell’s normal genome it won’t be replicated when the cell divides, it won’t be expressed, and in fact it will probably be broken down

37. FEATURES • It must be big enough to hold the gene to be cloned • It must be circular (or more accurately a closed loop), so that it is less likely to be broken down • It must contain control sequences,so that the gene will be replicated or expressed • It must contain marker genes, so that cells containing the vector can be identified

38. TYPES

39. PLASMID VECTORS • A plasmid is a DNA molecule that can replicate independently of the chromosomal DNA • Plasmids are circular molecules of DNA • Found in all three major domains: Archaea, Bacteria, and Eukarya • Plasmid vectors are used to clone DNA ranging in size from several base pairs to several thousands of base pairs (100bp -10kb).

40. BACTERIOPHAGE LAMBDA • Phage lambda is a bacteriophage or phage, i.e. bacterial virus, that uses E. coli as host. • Its structure is that of a typical phage: head, tail, tail fibres. • Lambda viral genome: 48.5 kb linear DNA with a 12 base ssDNA "sticky end" at both ends; these ends are complementary in sequence and can hybridize to each other (this is the cossite: cohesive ends). • Infection: lambda tail fibres adsorb to a cell surface receptor, the tail contracts, and the DNA is injected. • The DNA circularizes at the cossite, and lambda begins its life cycle in the E. coli host

41. TYPICAL CLONING EXPERIMENT THE CLONING OF DOLLY

42. Dolly is the first sheep created by a cloning expriment carried out by Wilmut and his colleagues at Roslin Institute, Scotland. • Cells from an adult Finn Dorset breed sheep’s mammary gland was removed. Finn Dorset is a pure white breed of sheep. • The cells were grown in a tissue culture. • The cells were starved of important nutrients. They stopped growing, dividing and became quiescent.

43. An oocyte was collected from a Scottish Blackface ewe (Ewes are female sheep) • The Scottish Blackface breed is a common breed of sheep in Scotland easily identified by its black face • The nucleus of the oocyte was removed while nucleus ‘donor’ collected from the quiescent mammary cell was injected in the enucleated oocyte

44. The nucleus was allowed to fuse with the cytoplasm of the enucleated oocyte and transferred into the reproductive chamber of a Blackface ewe • After 148 days, a normal length of time for the Finn Dorset breed of sheep, Dolly was born as a healthy, normal looking Finn Dorset • This proves that Dolly is not a product of a sneaky mating. This is because from the genetics of sheep breed, a Blackface breed can not produce a Finn Dorset breed

45. A DNA fingerprint was conducted which showed that Dolly’s DNA matched the cells from the tissue culture, not the cells from the ewe that gave birth to her.