How Do Genes Work?

Are Genes Composed of DNA or Protein? n Chromosomes, the known carriers of genes, are made of DNA and proteins n Proteins are more variable in structure than DNA, which was not thought to be sufficiently complex to code for all the operations of a cell. n DNA = a polymer made of 4 different types of nucleotides (adenine, cytosine, guanine, thymine) n Protein = a polymer made of 20 different types of amino acids

There are two strains of Streptococcus pneumoniae. ROUGH COLONY (R) SMOOTH COLONY (S) R strain is benign (Lacks a protective capsule, recognized and destroyed by host’s immune system) S strain is virulent (Polysaccharide capsule prevents detection by host’s immune system) Experiments showing DNA is the genetic material

Experiments by Griffiths, 1928 Conclusion:

Sample should Contain NO PROTEIN Add deoxy- ribonuclease Sample should Contain NO DNA Add ribonuclease Sample should Contain NO RNA Add proteinases Sample should contain NO PROTEIN Add DNAse Sample should contain NO DNA Add RNAse Sample should contain NO RNA Add R cells S cells appear S cells appear No S cells appear Transformation occurs No transformation occurs Conclusion: Transformation cannot occur unless DNA is present. Therefore, DNA must be the hereditary material. Add R cells Add proteinases Heat-killed S cells Lipids Carbohydrates 1. Remove lipids and carbohydrates from a solution of heat-killed S cells. Proteins, RNA, and DNA remain. 2. Treat solutions with enzymes to destroy proteins, RNA, or DNA. 3. Add a portion of each sample to a culture containing R cells. Observe if transformation occurs. Experiments by Avery, MacLeod and McCarty (1944)

Are Genes Composed of DNA or Protein? n Transformation experiments provide the first evidence that genes are DNA. Griffith: material from dead virulent Streptococcus can transform benign Streptococcus into a virulent strain. Avery et al.: extracted material from dead virulent Streptococcus and treated it with enzymes to destroy either DNA or RNA or proteins. Only DNA-destroying enzymes prevented transformation, hence DNA is the genetic material.

Are Genes Composed of DNA or Protein? n Not everyone was convinced n Hershey and Chase (1952): T2 virus experiments convinces skeptics that DNA is the genetic material since the material injected by the virus into host cells is DNA, not protein.

Bacteriophages are viruses that infect bacterial cells

Host cell genome Virus particle 1. Viral genome enters host cell. Host cell genome Virus particle 1. Viral genome enters host cell. 2. Viral genome is replicated and transcribed. DNA mRNA Host cell genome Virus particle 1. Viral genome enters host cell. 2. Viral genome is replicated and transcribed. DNA mRNA 3. Viral particles produced Protein New viruses released 4. Particles assembled inside host. How do viruses work?

Experiments by Hershey and Chase (1952) n Protein contains sulfur, but not phosphorous n DNA contains phosphorous, but not sulfur n Growing viruses with radioactive sulfur will label proteins but not DNA n Growing viruses with radioactive phosphorous will label DNA but not proteins

Experiments by Hershey and Chase (1952)

The structure and replication of genetic material n DNA structure was determined by James Watson and Francis Crick using data from Rosalind Franklin n In order to pass on genetic material from parent (cell) to offspring (cell), the genetic material must be duplicated: DNA replication One chromosome (Unreplicated state) One chromosome (Replicated state) Centromere Sister chromatids DNA replication

Figure 16.0 Watson and Crick

Figure 16.4 Rosalind Franklin and her X-ray diffraction photo of DNA

P P P CH 2 5' 3' O OH Base Structure of dNTPs Free DNA nucleotides are called dNTPs 1' 2' 4'

Review of DNA structure: DNA and RNA are polymers made of nucleotide subunits Nucleotide Ribose Deoxyribose PO O O–O– –O–O Phosphate group O N Nitrogenous base 5-carbon sugar OO HH HOH H H HO 5 CH 2 OH 2C2C HH H H N O H N NH 2 Pyrimidines NH O H N O NH 2 N N N H N Cytosine (C) Adenine (A) Uracil (U) Purines Thymine (T) Guanine (G) H3CH3C NH N H H N N NH 2 NH O N C3C3 4C4C 1C1C 2C2CC3C3 1C1C 4C4C HO 5 CH 2 O O

A single strand of nucleotides is made when a phosphodiester bond is formed between the 3’ C of one nucleotide and the 5’ C of another 5' 3'

DNA is double stranded with the 2 strands held together by hydrogen bonds between complementary bases 5´ 3´ 5´ 3´ 5´ T G C T T T C G A C T G T T G A C G A A G C G T A A C A C A T A G C G C A T TA C G G A T T A C G T A A T C G A T Cartoon of base pairing Cartoon of double helix Space-filling model of double helix 3´ 5´ 3´ 5´ DNA is a double helix.

Figure 16.6 Base pairing in DNA

Unnumbered Figure (page 292) Purine and pyridimine

Distinguishing between Models of DNA Replication n Three different models of how DNA might replicate were proposed based on DNA structure. Semi-conservative replication Conservative replication Dispersive replication

Figure 16.8 Three alternative models of DNA replication

Distinguishing Between Models of DNA Replication n The Meselsohn and Stahl experiment determines which model is correct. 15 N was fed to growing E. coli cells to mark DNA (“heavy” DNA), then cells were switched to 14 N. Newly synthesized DNA will incorporate 14 N (“light” DNA. 15 N 14 N 15 N 14 N

Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 1)

Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 2)

Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 3)

Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 4)