BIOL 360 - Module 1 - DNA Structure.pdf

Kwame Nkrumah University of Science & Technology, Kumasi, Ghana Module 1 S t r u c t u r e Prof. Matthew Glover Addo Start Module  Featured image Module 1 Module Dashboard...

Kwame Nkrumah University of Science & Technology, Kumasi, Ghana Module 1 S t r u c t u r e Prof. Matthew Glover Addo Start Module  Featured image Module 1 Module Dashboard FILTER DNA Structure Lessons Assignments 4 Start Learning 3 Dashboard hig Pick a Lesson Introduction Lesson 1 Lessons DNA Structure h1 t1 title Assignments Lesson 2 Denaturation & t2 Play h2 Renaturation Watch an overview Lesson 3 video on DNA t3 DNA Functions h3 2  Featured image Module Objectives FILTER Module 1 DNA Structure Dashboard 1 To know and appreciate the structure of DNA Introduction in terms of its components (i.e. nucleotides:- phosphate, pentose sugar, and a base) hig Lessons title Assignments 2 To know certain properties of DNA like denaturation, renaturation, bouyant density etc. 3  Featured image Module Lessons FILTER Module 1 DNA Structure Dashboard Introduction t1 t2 t3 Lessons hig Lesson 1 h1 Lesson 2 title h2 Lesson 3 h3 Assignments DNA Structure Denaturation & DNA Functions Learn about the discovery and components of DNA structure Renaturation Learn the important roles of DNA Start Start Start 4  Module 1 DNA Structure Lesson 1 Structure of Deoxyribonucleic t1 h1 hig Acid (DNA) Learn about the discovery and components of DNA structure t3 Learn Now t2 Module 1 DNA Structure Structure of Deoxyribonucleic Acid (DNA) t1 FILTER All experiments described so far point hig to nucleic acid (DNA or h1 RNA) as the carrier of genetic information 6 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure DNA Discovery t1 FILTER Early 1950’s hig DNA structure h1 elucidated Rosalind Maurice Erwin James Francis Chargaff Watson Franklin Wilkins Crick h2 Biochemist Molecular X-ray Crystallographer Biologist Learn More 7 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure DNA Discovery t1 FILTER This was achieved through effective investigation of scientists like: hig h1 Rosalind Franklin Maurice Wilkins Francis Crick James Watson Erwin Chargaff h2 h2 h2 h2 h2 X-ray X-ray Molecular Molecular Biochemist Crystallographer Crystallographer Biologist Biologist Learn More Learn More Learn More Learn More Learn More 8 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure DNA Discovery t1 FILTER Foundations on which the DNA structure were based: 1 Structural and Theoretical Chemistry Analysis of base compositions of DNA’s hig h1 from a variety of species 9 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure DNA Discovery t1 FILTER Foundations on which the DNA structure were based: 1 Structural and Theoretical Chemistry Analysis 2 X-ray Crystallographic hig h1 Data 10 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure DNA Discovery t1 FILTER 1Structural and Theoretical Foundations Chemistry on which the Analysis DNA structure 2 were based: X-ray Crystallographic Data 3 Structural Models hig The ability of Watson and Crick to build structural h1 models that were consistent with the chemical and physical data 11 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure DNA Discovery t1 FILTER Aaron Levene provided important information about the chemical hig composition of DNA h1 and concluded that, it is a polymer of purine and pyrimidine nucleotides 12 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure DNA Discovery t1 FILTER Using an x-ray crystallographic picture of DNA made by Franklin, Watson and Crick were able hig h1 to propose a model for the structure of DNA 13 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure DNA is a Double Helix t1 FILTER According to the model, DNA is composed of two long, unbranched polymers of deoxynucleotides lying hig h1 side by side 14 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure DNA is a Double Helix t1 FILTER It is a high molecular weight polymeric compound which consists of two molecules that are arranged into a ladder-like hig h1 structure called a Double Helix 15 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure DNA is a Double Helix t1 FILTER The backbone of the helix is composed of two chains with alternating sugar-phosphate units Alternating sugar-phosphate hig h1 Backbone units 16 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides t1 FILTER A molecule of DNA is made up hig of millions of tiny h1 subunits called Nucleotides 17 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides t1 FILTER Each nucleotide consists of: 1 Phosphate group hig Pentose sugar h1 2 3 Nitrogenous base 18 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides t1 FILTER Sugar Phosphate Backbone hig “Rungs” h1 Bases 19 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides t1 FILTER Complete hydrolysis of DNA yield hig h1 Phosphate Pentose Pyrimidine & Group Sugar Purine Bases 20 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Nitrogenous Bases Types t1 FILTER 1 Adenine 2 Thymine A T hig h1 3 Cytosine 4 Guanine C G 21 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Nitrogenous Bases Purines t1 FILTER Purines are heterocyclic Hypoxanthine hig ring of carbon h1 and nitrogen atoms Xanthine 22 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Nitrogenous Bases Pyrimides t1 FILTER Pyrimidines are cyclic ring of carbon and hig h1 nitrogen atoms Orotic Acid 23 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Nitrogenous Bases t1 FILTER Note The style of numbering of the hig pyrimidine ring in the purines differs h1 from that used for the pyrimidines themselves Adenine Cytosine 24 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Nitrogenous Bases Major Bases t1 FILTER Major bases Adenine Thymine found in (DNA only) DNA & hig h1 RNA 1 Guanine Cytosine Uracil 25 t3 t2 (RNA only)  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Nitrogenous Bases Major Bases t1 FILTER In certain bacterial viruses, cytosine is replaced by 5-methylcytosine or 5-hydromethylcytosine hig h1 Cytosine 5-methylcytosine 5-hydromethylcytosine 26 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Nitrogenous Bases Base Pairing t1 FILTER Each base will only bond with one other specific base Thymine (T) Cytosine (C) hig h1 Base Pair Adenine (A) Guanine (G) 27 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Nitrogenous Bases Base Pairing t1 FILTER hig h1 28 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Nitrogenous Bases Base Pairing t1 FILTER A T Because of this C G complementary base pairing, the order of T A the bases in one strand C G hig h1 determines the order A T of the bases in the G C other strand T A 29 t3 t2 C G  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Pentose Sugar t1 FILTER A Pentose sugar is a 5-carbon sugar in a ring form hig h1 30 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Pentose Sugar The Sugar Component t1 FILTER The sugar component of DNA is 2- hig h1 deoxyribose 2- 31 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Pentose Sugar The Sugar Component t1 FILTER The absence of OH- at carbon 2 has wide ranging effects on hig h1 both their chemistry and structure 2- 32 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Pentose Sugar The Sugar Component t1 FILTER The presence of the bulky hydroxyl group on the 2- position 1 Limits the range of possible secondary hig h1 structures available to the RNA molecule 33 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Pentose Sugar The Sugar Component t1 FILTER The presence of the bulky hydroxyl group on the 2- position 2 Makes it more susceptible to chemical and enzymatic hig h1 degradation 34 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Pentose Sugar Nitrogenous Base Link t1 FILTER 9 Glycosidic bond Pentose Sugar – Adenine from position 1 on Purine bond the pentose ring hig h1 to N9 of purines 1 35 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Pentose Sugar Nitrogenous Base Link t1 FILTER Cytosine 1 Glycosidic bond Pentose Sugar – from position 1 on Pyrimidine bond hig h1 the pentose ring to 1 N1 of pyrimidines 36 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Pentose Sugar Nitrogenous Base Link t1 FILTER Nucleotide Diagram hig h1 37 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Pentose Sugar Nitrogenous Base Link t1 FILTER Note To avoid ambiguity between the numbering systems of Base hig the heterocyclic rings h1 and the sugar, positions on the pentose are given a prime (‘) 38 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Phosphate Group t1 FILTER HPO42- hig h1 39 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Phosphate Group t1 FILTER 5’ The 5’ position of one 3’ pentose ring is connected hig to the 3’ position of the h1 next pentose via a phosphate 5’ 40 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Phosphate Group t1 FILTER Thus, the sugar - phosphate backbone is hig said to consist of 5’ – 3’ h1 phosphodiester bond or linkages 41 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Phosphate Group t1 FILTER 5’ 3’ 5’ 3’ A C T C A G T C G C A T C hig h1 T G A G T C A G C G T A G 3’ 5’ 42 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Phosphate Group t1 FILTER When DNA (RNA) is broken into its constituent nucleotides, the cleavage may take place on either hig h1 side of the phosphodiester bonds 43 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Phosphate Group t1 FILTER 2 types of nucleotides released from N.A. 1 2 Base Base 5’ 3’ hig h1 Nucleoside-5’- Nucleoside-3’- 44 monophosphate monophosphate t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Phosphate Group t1 FILTER All the nucleotides can exist in a form in which there is 5’ hig more than one h1 phosphate group linked to the 5’ position Nucleoside-5’-triophosphate 45 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Phosphate Group t1 FILTER hig h1 Adenosine-5-triophosphate 46 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Phosphate Group t1 FILTER Energy-rich Bonds used to provide an energy source for various cellular activities    hig h1 47 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Phosphate Group t1 FILTER Partial hydrolysis yields nucleosides and nucleotides compounds hig h1 Nucleoside Nucleotide 48 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Phosphate Group t1 FILTER Nucleoside Nucleotide A base linked to a sugar When a phosphate group is added to a base-sugar hig h1 Phosphate Base Base group 49 t3 t2 Sugar Sugar  Lesson 1 Structure of DNA t1 FILTER Module 1 Lesson Assignment 1 DNA Structure Assignment 1 Assignment 2 Assignment 3 Assignment 4 Dashboard Introduction Bases, nucleosides, and nucleotides have Lessons related names. Learn these nomenclature Assignments hig depending on the base present. h1 t i t l e Example: adenine- adenosine-adenylic acid-dAMP 50 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Shorthand Notation t1 FILTER The representation of polynucleotide chains by complete formulae is clumsy and therefore has hig h1 become necessary to use schematic systems 51 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Nucleotides Shorthand Notation t1 FILTER 5’ → 3’ polarity on the top line of the sequence with the complementary strand of opposite polarity lying below hig h1 5’- GTCAG - 3’ 3’- CAGTC - 5’ 52 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure The Double-Helix Some Characteristics t1 FILTER 1 Helix Regular Helix has complete turn (pitch) of 34Å or (3.4nm) Helix 3.4nm hig h1 53 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure The Double-Helix Some Characteristics t1 FILTER 1 Helix = 3.4nm Diameter 2nm 2 Diameter Has a diameter of ≈ 20Å or (2nm) hig h1 54 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure The Double-Helix Some Characteristics t1 FILTER 1 Helix = 3.4nm 2 Diameter = 2nm Adjacent Nucleotides 3 Nucleotides Distance 0.34nm hig The distance between h1 adjacent nucleotides is 3.4Å or (0.34nm) 55 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure The Double-Helix Some Characteristics t1 FILTER 1 Helix = 3.4nm 2 Diameter = 2nm 10 Nucleotides Per turn 3 Nucleotides Distance = 0.34nm 4 Nucleotides Per Turn hig h1 There must be 10 nucleotides per turn 56 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure The Double-Helix Some Characteristics t1 FILTER 1 Helix = 3.4nm 2 Polynucleotide 2 Diameter = 2nm chains 3 Nucleotides Distance = 0.34nm 4 Nucleotides Per Turn = 10 hig h1 5 Polynucleotide Chains i The density of DNA suggests that, the helix must contain 2 polynucleotide chains 57 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure The Double-Helix Some Characteristics t1 FILTER 1 Helix = 3.4nm 5’ 3’ 2 Polynucleotide Diameter = 2nm Chains 3 The two chains Nucleotides are=anti- Distance 0.34nm parallel which means if one 4 strand hasPer Nucleotides 5’→3’ Turnpolarity = 10 from hig h1 i top to bottom, then, the other 5 must have 3’→5’ polarity from 5’ The 3’ topdensity of DNA suggests to bottom that, the helix must contain 2 polynucleotide chains 58 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure The Double-Helix Some Characteristics t1 FILTER 1 = 3.4nm Helix 2= 2nm Diameter 3 Nucleotides = 0.34nm Distance 4 Nucleotides = 10Per Turn 5 Polynucleotide Chains = 2 i 6 Proportion of Bases hig Irrespective of the actual h1 amounts of each base, the proportion of G is always the same as the proportion of C in DNA, and that of A is always the same as T 59 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure The Double-Helix Some Characteristics t1 FILTER 1 Helix = 3.4nm 2 Diameter = 2nm 3 Nucleotides Distance = 0.34nm hig h1 4 Nucleotides Per Turn = 10 5 Polynucleotide Chains = 2 6 Proportion of Bases = G=C, A=T 60 t3 t2  Lesson 1 Structure of DNA t1 FILTER Module 1 Lesson Assignment 2 DNA Structure Assignment 1 Assignment 2 Assignment 3 Assignment 4 Dashboard Read on Chargaff’s Rule Introduction Lessons t h1 i t l e Assignments hig 61 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure The Double-Helix Contributions of the Base Pairs t1 FILTER The base pairs affect the thermodynamic stability of the double helix in two ways: hig 1 Hydrogen Bonding h1 2 Hydrophobic base-stacking 62 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure The Double-Helix Contributions of the Base Pairs t1 FILTER 1 Hydrogen Bonding between the bases in each pair releases energy hig corresponding to 3 H- h1 bonds per G-C and 2H- bonds per A - T pair 63 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure The Double-Helix Contributions of the Base Pairs t1 FILTER 1 Hydrogen Bonding Due to the increased number of hydrogen hig bonds holding together h1 a G:C nucleotide pair, regions of DNA rich in G + C are more stable than regions rich in A + T. 64 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure The Double-Helix Contributions of the Base Pairs t1 FILTER 1 Hydrogen Bonding On denaturation, the A + T-rich regions will hig melt first h1 65 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure The Double-Helix Contributions of the Base Pairs t1 FILTER 1 Hydrogen Bonding 2 Hydrophobic base- hig stacking The interaction between h1 the electron systems of the base pairs also results in hydrophobic base-stacking 66 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Different Forms of DNA t1 FILTER Although the basic model put forward by Watson and Crick hig remains close to the accepted structure of h1 the DNA molecule in solution, varieties exist 67 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Different Forms of DNA t1 FILTER Refined X-ray studies show a variety of possible structures like A-, hig B-, C-, and Z- h1 forms, depending on the conditions chosen to produce the DNA 68 t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Different Forms of DNA t1 FILTER A DNA 1.2 nm Minor groove hig h1 2.8 nm Major groove 11 Bp/turn +34.7o Rotation/Bp 69 A DNA t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Different Forms of DNA t1 FILTER B DNA Minor 1 nm groove hig h1 3.9 nm Major 10.4 Bp/turn groove +34.6o Rotation/Bp 70 B DNA t3 t2  Lesson 1 Structure of DNA Module 1 DNA Structure Different Forms of DNA t1 FILTER Z DNA 0.9 nm hig h1 6.8 nm 12 Bp/turn -30.0o Rotation/Bp 71 Z DNA t3 t2  Lesson 1 Structure of DNA t1 FILTER Module 1 Lesson Assignment 3 DNA Structure Assignment 1 Assignment 2 Assignment 3 Assignment 4 Dashboard Read and summarize the different forms of DNA Introduction under the following headings: 1. Relative Humidity Lessons 2. Pitch in nm 3. Residues per turn and t h1 i t l e Assignments hig 4. Inclination of b.p. from horizontal Example: The B-form under R.H of 92% has a pitch of 3.4nm, 10 bases per turn and has 0o angle of 72 inclination to the horizontal. t3 t2  Featured image Module Lessons FILTER Module 1 DNA Structure Dashboard Introduction t1 t2 t3 Lessons hig Lesson 1 h1 Lesson 2 title h2 Lesson 3 h3 Assignments DNA Structure Denaturation & DNA Functions Learn about the discovery and components of DNA structure Renaturation Learn the important roles of DNA Start Start Start 73  Module 1 DNA Structure Lesson 2 DNA Denaturation t2 h2 & Renaturation hig Learn how DNA changes from a double helix t3 to a single stranded DNA random coil Learn Now t1  Lesson 2 DNA Denaturation and Renaturation Module 1 DNA Structure t2 FILTER When double-stranded DNA molecules are subjected to extremes of temperature or pH, the hydrogen bonds of the hig h2 double helix are rupture and the two strands no longer hold together 75 t3 t1  Lesson 2 DNA Denaturation and Renaturation Module 1 DNA Structure t2 FILTER The DNA is said to denature and changes from a double helix to a single stranded DNA random coil hig h2 76 t3 t1  Lesson 2 DNA Denaturation and Renaturation Module 1 DNA Structure t2 FILTER When heat is used as the denaturant, the DNA is said to melt and the temperature hig at which the strands h2 separate is the melting temperature or transition temperature (Tm) 77 t3 t1  Lesson 2 DNA Denaturation and Renaturation Module 1 DNA Structure t2 FILTER When duplex DNA melts, the hydrogen bonds break and the bases unstack with the consequence hig h2 that the absorption at 260 nm rises by 30-40% (20-30%) 78 t3 t1  Lesson 2 DNA Denaturation and Renaturation Module 1 DNA Structure t2 FILTER This rise in absorption is the hyperchromic hig effect or shift h2 and is used to monitor the melting of DNA 79 t3 t1  Lesson 2 DNA Denaturation and Renaturation Module 1 DNA Structure t2 FILTER The nature of the melting transition is affected by several factors: 1 G + C Content hig 2 The nature of the solvent h2 3 The nature of the DNA 80 t3 t1  Lesson 2 DNA Denaturation and Renaturation Module 1 DNA Structure t2 FILTER 1 G + C Content The higher the G+C content of DNA, the more stable the molecule will be and hig h2 hence, the higher the melting temperature 81 t3 t1  Lesson 2 DNA Denaturation and Renaturation Module 1 DNA Structure t2 FILTER Denaturation by heat of hig DNAs from different h2 organisms 82 t3 t1  Lesson 2 DNA Denaturation & Renaturation t2 FILTER Module 1 Lesson Assignment 4 DNA Structure Assignment 1 Assignment 2 Assignment 3 Assignment 4 Dashboard Read and make notes on the other factors Introduction Lessons 2 The nature of the solvent The nature of the DNA t h2 i t l e Assignments hig 3 83 t3 t1  Featured image Module Lessons FILTER Module 1 DNA Structure Dashboard Introduction t1 t2 t3 Lessons hig Lesson 1 h1 Lesson 2 title h2 Lesson 3 h3 Assignments DNA Structure Denaturation & DNA Functions Learn about the discovery and components of DNA structure Renaturation Learn the important roles of DNA Start Start Start 84  Module 1 DNA Structure t3 h3 hig Lesson 3 DNA Functions Learn the important roles of DNA t2 Learn Now t1  Lesson 3 DNA Functions Module 1 DNA Structure t3 FILTER 1 Storage of genetic information 2 Self-duplication (replication) & hig h3 inheritance 3 Expression of the genetic message 86 t2 t1  Lesson 3 DNA Functions Module 1 DNA Structure t3 FILTER 1 Storage of genetic information 2 Self-duplication (replication) & hig h3 inheritance 3 Expression of the genetic message 87 t2 t1  Lesson 3 DNA Functions Module 1 DNA Structure t3 FILTER 1 Storage of genetic information 2 Self-duplication (replication) & hig h3 inheritance 3 Expression of the genetic message 88 t2 t1  Lesson 3 DNA Functions Major Function Module 1 DNA Structure t3 FILTER DNA’s major function: hig Code for h3 Proteins 89 t2 t1  Lesson 3 Module 1 The Central Dogma of Gene Expression DNA Structure t3 FILTER hig h3 90 t2 t1  Featured image Module Lessons FILTER Module 1 DNA Structure Dashboard Introduction t1 t2 t3 Lessons hig Lesson 1 h1 Lesson 2 title h2 Lesson 3 h3 Assignments DNA Structure Denaturation & DNA Functions Learn about the discovery and components of DNA structure Renaturation Learn the important roles of DNA Start Start Start 91  Module 1 DNA Structure hig End of Module 1 t3 t2 End Class Next Module t1