Molecular Biology (BIO1004/2001)

1. Molecular Biology(BIO1004/2001) Kathleen Lobban Kathleenlobban@yahoo.com

2. MODULE CONTENT • Structure of DNA • DNA Replication • Transcription and Protein synthesis • Molecular Biology Techniques • Recombinant DNA technology • Gene Expression and Regulation in Prokaryotes and Eukaryotes

3. ASSESSMENT PROCEDURES • Test 1 (Week of October 12, 2009 ) 15% • Test 2 (Week of November 16, 2009 ) 15% • Practicals 10% • Project (Week of October 26, 2009 ) 10% • Final Exam (Week of October 12, 2009 ) 50%

4. UNIT 1 Structure of DNA

5. DNA • Deoxyribonucleic acid • Belongs to the class of macromolecules called nucleic acids. • The other nucleic acid is RNA • (ribonucleic acid)

6. Composition of DNA • DNA is a polymer. • The monomer units of DNA are nucleotides • Therefore the polymer is known as a "polynucleotide." • Each nucleotide consists of • a 5-carbon sugar (deoxyribose) • a nitrogen-containing base • a phosphategroup.

7. Composition of DNA

8. Nitrogenous Bases

9. The Nucleotides Base Nucleoside Nucleotide (dNTP) Adenine adenosine adenosine triphosphate ATP / dATP Guanine guanosine guanosine triphosphate GTP / dGTP Cytosine cytidine cytidine triphosphate CTP / dCTP Uracil uridine uridine triphosphate UTP /dUTP Thymine thymidine thymidine triphosphate TTP / dTTP

10. Other Functions of Nucleotides • They carry chemical in their bonds which can be easily released for use by the cell eg ATP and GTP • They combine with other group to form coenzymes: eg coenzyme A • They are used as specific signaling molecules within cells, eg. Cyclic AMP (cAMP) serves to signal switching on of the lac operon in prokaryotes.

11. Composition of DNA Base Pairs • Adenine forms 2 hydrogen bonds with Thymine on the opposite strand • Adenine (A) will only bond with Thymine (T) • Guanine forms 3 hydrogen bonds with Cytosine on the opposite strand. • Guanine (G) will only bond with Cytosine (C).

14. DNA molecule • First described by James D. Watson and Francis Crick in 1953. • In a DNA molecule, the two strands are not parallel, but intertwined with each other.  • The two strands form a "double helix" structure

15. DNA molecule • The DNA backbone is an alternating sugar-phosphate sequence. • The deoxyribose sugars are joined at both the 3'- and 5'-carbon to phosphate groups by phosphodiester bonds.

16. DNA molecule • Chain has a direction (polarity) • 5΄ end - phosphate • 3΄ end - hydroxyl

17. DNA molecule The two polynucleotide chains run in opposite directions - antiparallel

18. The DNA Double Helix • Two DNA strands form a right-handed helical spiral • The sugar-phosphate backbones of the two DNA strands wind around the helix axis like the railing of a spiral staircase • The bases of the individual nucleotides are on the inside of the helix, stacked on top of each other like the steps of a spiral staircase

19. DNA molecule • The sugar-phosphate backbone of DNA is polar, and therefore hydrophilic. • The bases, are relatively non-polar and therefore hydrophobic. • These hydrostatic forces have a very stabilizing effect on the overall structure of the DNA double helix • Therefore there is a strong pressure holding the two strands of DNA together.

20. The DNA Double Helix • The helix makes a turn every 3.4 nm • The distance between two neighboring base pairs is 0.34 nm.  • Hence, there are about 10 pairs per turn.  • The intertwined strands make two grooves of different widths • The major groove facilitate binding with specific proteins. • The minor groove

23. Watson and Crick’s Model of DNA In 1928 Frederick Griffith was working on a project that enabled others to point out that DNA was the molecule of inheritance.  • The experiment involved mice and bacteria that cause pneumonia - a virulent and a non-virulent kind.  • He injected the virulent into a mouse and the mouse died.  • Next he injected the non-virulent into a mouse and the mouse lived.  • After this, he heated up the virulent strain to kill it and then injected it into a mouse.  The mouse lived.  • Last he injected non-virulent pneumonia and virulent pneumonia, that had been heated and killed, into a mouse.  This mouse died.    

24. Watson and Crick’s Model of DNA • Why?  Griffith thought that the killed virulent bacteria had passed on a characteristic to the non-virulent one to make it virulent - TRANSFORMATION.  He thought that this characteristic was in the “inheritance molecule”. 

26. Watson and Crick’s Model of DNA In 1942 Oswald Avery • continued with Griffith’s experiment to see what the inheritance molecule was.  • He destroyed the lipids, ribonucleic acids, carbohydrates, and proteins of the virulent pneumonia.  Transformation still occurred after this.  • Next he destroyed the deoxyribonucleic acid.  Transformation did not occur.  Avery had found the inheritance molecule, DNA!

27. Watson and Crick’s Model of DNA 1940’s Erwin Chargaff •   noticed a pattern in the amounts of the four bases: adenine, guanine, cytosine, and thymine.  • In samples of DNA from different organisms : • the amount of adenine = to the amount of thymine • the amount of guanine = to the amount of cytosine.  • This discovery later became Chargaff’s Rule.

28. Watson and Crick’s Model of DNA Rosalind Franklin and Maurice Wilkins • Decided to try to make a crystal of the DNA molecule.  They obtained an x-ray pattern.  • The pattern appeared to contain rungs, like those on a ladder between to strands that are side by side.  It also showed by an “X” shape that DNA had a helix shape. www.who2.com/.../watson-v-franklin-round-27.html

29. Watson and Crick’s Model of DNA • In 1953 James Watson and Francis Crick • saw Franklin and Wilkin's picture of the X-ray and • made an accurate model - a double helix with little rungs connecting the two strands • But how to bond the bases together ? • How to solve the problem of the sizes of the bases?  • Adenine and Guanine were purines having two carbon-nitrogen rings in their structures.  • Thymine and Cytosine were pyrimidines having one carbon-nitrogen ring in its structure. • If purines and the pyrimidines were together, then DNA would look wobly and crooked.  • If they paired Thymine with Adenine and Guanine with Cytosine, DNA would look uniform (Chargaff's rule)  • Each side is a complete compliment of the other.

31. Watson and Crick Model of DNA • http://www.johnkyrk.com/DNAanatomy.html

32. Remembering Rosalind • By using the picture of the crystallized DNA, Watson and Crick were able to put together the model of DNA.  •   Watson and Crick used information from Avery, Chargaff, Griffith, and others.  • They simply pieced together the puzzle.  • The Nobel Prize was awarded to Watson, Crick, and Maurice Wilkins.  • Rosalind Franklin did not receive the prize because she had died of cancer by this time.  Maurice Wilkins was able to share the prize with Watson and Crick, though, because of his work with Franklin.  Her accomplishment should never be forgotten. 

33. Forms of DNA • DNA exists in many possible conformations. • only A-DNA, B-DNA, and Z-DNA have been observed in cells. • conformation depends on: • the DNA sequence • the amount and direction of supercoiling • chemical modifications of the bases • solution conditions (eg concentration of metal ions, salt concentration and level of hydration).

34. B -Form of DNA • Most common under the conditions found in cells. • Has a major and minor groove • Has 10 base pairs per turn • One turn spans 3.4 nm

35. A -Form of DNA • With higher salt concentrations or with alcohol added, the DNA structure may change to A form • A wider right-handed spiral • A shallow, wider minor groove • A narrower, deeper major groove • Has 11 bases per turn • One turns spans 2.3 nm

37. Z -Form of DNA • The DNA molecule with alternating G-C sequences in alcohol or high salt solution tends to have such structure • Bases seem to zigzag • The strands turn about the helical axis in a left-handed spiral. • Has a narrow deep groove • Has 12 bases per turn. • One turn spans 4.6 nm

38. A – DNA B-DNA Z-DNA

39. Chromosomes • Chromosomes – highly coiled condensed packages of DNA • Present in both eukaryotes and prokaryotes • Eukaryotes – linear • Prokaryotes – most closed circular The human genome has 3,000,000,000 base pairs packed into 23 chromosomes!

41. Organization of Eukaryotic Genomes • DNA is packed into chromosomes with the help of proteins - histones. • The histones associate with DNA forming structures called nucleosomes. • Each nucleosome complex consists of a beadlike structure with 146 base pairs of DNA wrapped around a disc-shaped core of eight histone molecules.

42. Nucleosome

43. Chromosomes • Nucleosomes are 11nm in diameter. • The packed nucleosome state occurs when a ninth histone called H1, associates with the linker DNA packing adjacent nucleosomes together forming a 30nm diameter thread. • In the extended chromosome , these 30nm diameter threads form large coiled loops held together by a set of non-histone scaffolding proteins.

45. Eukaryotic Genomes • organized into multiple chromosomes - varying in size and numbers depending on the species: • Human cells haploid 23 • Fruit flies haploid 4 • Yeast haploid 16 • Cats haploid 19 • Dogs haploid 39

47. Bacterial Genome Organization • Most have covalently closed, circular chromosomes and plasmids • Not all bacteria have a single circular chromosome: • some bacteria have multiple circular chromosomes • many bacteria have linear chromosomes and linear plasmids.

48. Bacterial Genome • The genetic material of a bacterium lies in the cytoplasm and is not surrounded by a nuclear envelope. • In most species it is contained in a single circular DNA molecule. • If stretched out to its full length, this molecule would be 1000 times longer than the cell itself. • Unlike eukaryotic chromosomes, the bacterial DNA has little proteins associated with it.

49. Plasmids • In addition to the genomic DNA, most bacteria have a small amount of genetic information present as one or more plasmids – extrachromosomal circular DNA. • Plasmids can replicate independently of the genomic DNA or become integrated into it. • Bacterial plasmids frequently have genes that code for: • catabolic enzymes • genetic exchange • resistance to antibiotic.