THE CHEMICAL NATURE OF THE GENE MOLECULAR GENETICS THE CHEMICAL NATURE OF THE GENE © 2016 Paul Billiet ODWS

What does a gene do? The gene must be able to hold information and decode it (translate it) into an organism as it grows and develops It must be able to copy itself so that it can be passed on to future generations © 2016 Paul Billiet ODWS

What does a gene look like? It must be a big molecule to hold the large amount of information required to build an organism It must be a complex molecule to provide the necessary variation to code the instructions that control growth and development © 2016 Paul Billiet ODWS

Four classes of molecules which could form genes Biological macromolecules Elements Building Blocks Polysaccharides (carbohydrates) CHO Monosaccharides Lipids (Fats, oils and waxes) CHO Fatty acids (and glycerol) Polypeptides (proteins) CHONS Amino acids Polynucleotides (Nucleic acids) CHONP Nucleotides © 2016 Paul Billiet ODWS

Griffiths (1928) Tried to determine what genetic material was made of. profiles.nlm.nih.gov/CC/A/A/B/N/_/ccaabn_.jpg © 2016 Paul Billiet ODWS

Griffiths’ Experiment Pneumococcus bacteria on mice 2 STRAINS S-type Smooth colonies Virulent R-type Rough colonies Avirulent Innoculate into mice Dead from pneumonia Not killed © 2016 Paul Billiet ODWS

Griffiths’ Experiment Live R-type (harmless) + Heat-killed S-type CONTROL Live R-type only CONTROL Heat-killed S-type only Mice died from pneumonia No mice died No mice died Further test: Cultured lung fluid Live S-type found © 2016 Paul Billiet ODWS

Conclusion Transformation of R-type to S-type Transformation was brought about by some heat stable compound present in the dead S-type cells Called the TRANSFORMING PRINCIPLE © 2016 Paul Billiet ODWS

Avery, MacCleod & McCarthy (1944) Tried purifying the transforming principle to change R-type Pneumococcus to S-type © 2016 Paul Billiet ODWS

Results Conclusion The compound that had the most effect was: Colourless, viscous and heat stable It contains phosphorus It was not affected by trypsin (a protease) or amylase. It was not inhibited by RNAase It was inhibited by DNAase Conclusion The transforming principle is a DNA © 2016 Paul Billiet ODWS

Experiment Live R-type + DNA extracted and purified from S-type bacteria Experiment Mice died from pneumonia Live S-type bacteria cultured from the lung fluid These S-type bacteria remained virulent for generation after generation © 2016 Paul Billiet ODWS

Conclusion DNA is the transforming principle and it is hereditary material Criticism The DNA was not totally pure It was contaminated by a small amount of protein This protein could be the real transforming principle BUT Purer extracts of DNA were better at transforming the bacteria types. © 2016 Paul Billiet ODWS

Hershey & Chase (1952) To determine the nature of hereditary material used by viruses © 2016 Paul Billiet ODWS

Virus A virulant sub-microscopic agent which will pass through filters that normally stop bacteria. Ebola virus © 2016 Paul Billiet ODWS

Virus Luria (1955) “bits of heredity in search of chromosomes” Lwoff (1966) “viruses are viruses because they are viruses”. © 2016 Paul Billiet ODWS

Virus Three properties of all viruses Infectivity Absence of biochemical apparatus (no metabolism). A virus is not alive Possess only one type of nucleic acid (DNA or RNA). © 2016 Paul Billiet ODWS

Types of virus Positive RNA viruses: TMV & Enterovirus (polio) RNA used like mRNA directly by the host cell for translation. Negative RNA viruses: Ebolavirus, Orthomyxoviridae (influenza) Viral RNA is transcribed into positive RNA before it is translated. The RNA polymerase that does this is supplied by the virus. Retrovirus: HIV Inject RNA strand plus reverse transcriptase. A DNA copy is made and used for the expression of viral genes. Bacteriophages: DNA viruses. © 2016 Paul Billiet ODWS

Virus structure Composition: Protein and nucleic acid Organisation : Protein coat covering a nucleic acid core A virus does not posses a cellular structure Crystallisable. © 2016 Paul Billiet ODWS

PHAGE ATTACK! © 2016 Paul Billiet ODWS

PHAGE ATTACK! © 2016 Paul Billiet ODWS T2 Phage particle

E. coli bacterium before infection © 2016 Paul Billiet ODWS

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Virus “life” cycle Lysis Packaging Infection Protein synthesis Replication © 2016 Paul Billiet ODWS

The Herschey & Chase Experiment Proteins: CHONS labeled with 35S Nucleic acids CHONP labeled with 32P Phage particle © 2016 Paul Billiet ODWS

Milk shakes at Cold Spring Harbour The Waring Blender © 2016 Paul Billiet ODWS

The Herschey & Chase Experiment Two types of T2 phages prepared: 35S labeled (proteins labeled) 32P labeled (nucleic acids labeled) Infect fresh E. coli host cells with each type Centrifuge to remove the surplus phages Agitate in the blender to remove phages coats from host cells Centrifuge again and collect: (a) phage coats (b) bacterial cells. © 2016 Paul Billiet ODWS

Destination of the radioactivity / % Results Centrifuge fraction Destination of the radioactivity / % 32P labeled phages 35S labeled phages Bacterial host cells 80 20 Phages © 2016 Paul Billiet ODWS

Observation Most of the 32P (i.e. DNA) is injected into the host but not much 35S (i.e. protein). © 2016 Paul Billiet ODWS

Error margin The 20% 35P in the phage coat fraction phages that had not been removed by the first centrifugation plus attached phages that had not yet injected their DNA The 20% 35S in the bacterial cell fraction could be due to phage coats which had not been successfully knocked off by the blender. © 2016 Paul Billiet ODWS

Going further Of the 32P which got into the host cells, half of it was found in phages produced from infected cells Less than 1% of the 35S appeared in the next generation viruses. Phage infecting a bacterium © 2016 Paul Billiet ODWS