B. Molecular Biology: 1. Chemical Nature of Genes and DNA Structure Dr Elizabeth Ellis BM102 Molecular Bioscience

Objectives By the end of this lecture you should be able to: Explain how the chemical nature of genes was determined experimentally by the work of Griffith, Avery and Hershey and Chase. Distinguish between a nucleoside, nucleotide and a polynucleotide Explain how a phosphodiester bond forms Understand the significance of complementary base pairing in DNA

Determining the chemical nature of genes The work of Mendel ( ) allowed him to discover the ‘particulate’ or ‘discrete’ nature of inheritance. And in 1911, Johannsen showed a link between genotype and phenotype But what is the molecular basis of inheritance? What are genes made of? Are they protein? Or DNA? Or RNA? Or something else? Three classic experiments: The Griffith Experiment (1928) The Avery Experiments (1944) The Hershey-Chase Experiments (1950s)

The Griffith Experiment Looked at the bacteria Streptococcus pneumoniae –Smooth strain (‘S’ form) –Rough strain (‘R’ form) 2 different phenotypes The characteristic was inherited

The Griffiths Experiment: background S form –Forms a capsule of carbohydrate –Capsule protects against host’s defenses –Virulent ie can survive and grow in the host R form –Unable to make the capsule –Non-virulent (Non-infective); host’s immune system can attack and destroy the bacteria

The Griffiths Experiment The heat killed S strain was able to TRANSFORM (ie change) the nonvirulent R strain

Avery, MacLeod, McCarty Experiment: What is the transforming factor? Proteases do not destroy the transforming factor –So, not protein Nucleases DO destroy the transforming factor So, transformation likely brought about by ‘DNA’

Hershey-Chase Experiment Bacteriophage or ‘phage These are viruses that infect and replicate in bacteria They are made of protein and DNA

Bacteriophage are known to attach to bacterial cells This attachment allows new bacteriophage to be produced within the cell How are new bacteriophage produced? What is the heritable material of the bacteriophage? Is it DNA or protein?

Hershey-Chase Experiment Proteins contain sulphur but DNA does not. Protein can be labelled with an isotope 35 Sulphur This is radioactive and can be detected

Hershey-Chase Experiment 1. Coat protein labelled with 35 S Results: Bacterial cell pellet containing new phage particles was NOT radioactive Conclusion: Protein is NOT passed into the bacterial cell or new phage so unlikely to be the heritable material

Hershey-Chase Experiment DNA contains phosphate but coat proteins do not DNA can be labelled with radioactive 32 Phosphate

Hershey-Chase Experiment 2. DNA labelled with 32 P Results: Bacterial cell pellet containing new phage particles was radioactive Conclusion: Radioactive DNA is passed into the bacterial cell and the new phage particles - and likely to be the heritable material

Conclusions: DNA is the heritable material in bacteria and bacteriophage: –It is not destroyed by proteases –It is destroyed by nucleases –Radioactive DNA gets passed on to next generation of bacteriophage

DNA structure DNA consists of four types of nucleotides Revision Dr Ferro’s lecture on nucleic acids Represented by the letters: A G T C

Base (eg: adenine) Nucleoside (eg: adenosine) Nucleotide (eg: adenosine mono phosphate) = base + sugar + phosphate

Purine and Pyrimidine Bases 2 rings 1 ring

DNA is a polynucleotide chain Chains of any length can be built up Each DNA chain is a specific sequence made up of the 4 different nucleotides A, T, G or C

Phosphodiester bond This bond forms by a condensation reaction, between the phosphate group of one nucleotide and the hydroxyl group attached to the carbon at position 3 in the second nucleotide 5’ phosphate 3’ hydroxyl Nucleotides are connected to each other by a phosphodiester bond

Phosphodiester Bond The phosphate ester links the 3’ and 5’ oxygens of the two sugar groups 3 5

Each DNA strand has two DISTINCT ends 5’ phosphate end 3’ hydroxyl end

In many organisms DNA is double-stranded = Double helix Two chains of DNA which are ANTIPARALLEL Run in OPPOSITE directions

Rosalind FranklinDNA diffraction

James Watson and Francis Crick Maurice Wilkins

Formation of double helix Hydrogen bonds are formed between the bases from opposite DNA chains. –A can only pair with T –G can only pair with C This is known as BASE-PAIRING The base-pairing rule supports an earlier observation made by Chargaff that the number of A’s in DNA equals the number of T’s

Hydrogen Bonding in Nucleic Acid Structure

Hydrogen Bonding in DNA Structure antiparallel

Which is stronger? A.A-T pair? B.G-C pair?

Complementarity If we know sequence of one strand of DNA, we can work out the sequence of the other strand Eg What is the complementary sequence to the following DNA sequence? 5’ A C G T A C T A G C G C G A A T 3’ NB Always include the 5’ and 3’ to show which end is which 3’ T G C A T G A T C G C G C T T A 5’ Top strand Bottom strand

Ratio of nucleotides varies in different species Different species have different proportions of each nucleotide ie some have more Gs (and hence Cs) and less A’s (and hence T’s) First described by Chargaff This is represented as the %GC content eg 40% G+C - so must also be 60% A+T Also, if an organism has 15% G, then must be 15% C Meaning the remaining 70% is (A+T), so 35% A and 35% T

Question If the proportion of adenine bases in DNA isolated from a sea urchin is 23%, what is the proportion of cytosine bases? A. 23% B. 46% C. 27% D. 54%

Summary DNA was shown by different experiments to be the heritable material DNA is a double helix made of two polynucleotide chains Each chain has a sugar-phosphate backbone on the outside with organic bases on the inside The two chains are held together by complementary base pairing The chains are antiparallel –The 5’ end of one chain lies next to the 3’ end of the other chain

Reading Brooker – Chapter 11