PowerLecture: Chapter 13 DNA Structure and Function

13.1 The Hunt Originally believed to be an unknown class of proteins Originally believed to be an unknown class of proteins Thinking was Thinking was Heritable traits are diverse Heritable traits are diverse Molecules encoding traits must be diverse Molecules encoding traits must be diverse Proteins are made of 20 amino acids and are structurally diverse Proteins are made of 20 amino acids and are structurally diverse

Miescher Discovered DNA Johann Miescher investigated the chemical composition of the nucleus Johann Miescher investigated the chemical composition of the nucleus Isolated an organic acid that was high in phosphorus Isolated an organic acid that was high in phosphorus He called it nuclein He called it nuclein

Griffith Discovers Transformation Attempting to develop a vaccine Attempting to develop a vaccine Isolated two strains of Streptococcus pneumoniae Isolated two strains of Streptococcus pneumoniae Rough strain was harmless Rough strain was harmless Smooth strain was pathogenic Smooth strain was pathogenic

Transformation What happened in the fourth experiment? What happened in the fourth experiment? The harmless R cells had been transformed by material from the dead S cells The harmless R cells had been transformed by material from the dead S cells Descendents of the transformed cells were also pathogenic Descendents of the transformed cells were also pathogenic

Avery, McCarty, and MacLeod Repeated Griffith’s Experiment Oswald Avery Maclyn McCarty Colin MacLeod

Avery, McCarty, and MacLeod Added the non-deadly Rough Type of Bacteria to the Heat-Killed Smooth TypeCarbohydratesLipidsProteinsRNADNA To the Heat-Killed Smooth Type, added enzymes that destroyed…

S-Type Carbohydrates Destroyed S-Type Lipids Destroyed S-Type Proteins Destroyed S-Type RNA Destroyed S-Type DNA Destroyed Conclusion: DNA was the transforming factor!

Oswald & Avery What is the transforming material? What is the transforming material? Cell extracts treated with protein-digesting enzymes could still transform bacteria Cell extracts treated with protein-digesting enzymes could still transform bacteria Cell extracts treated with DNA-digesting enzymes lost their transforming ability Cell extracts treated with DNA-digesting enzymes lost their transforming ability Concluded that DNA, not protein, transforms bacteria Concluded that DNA, not protein, transforms bacteria

The Hershey-Chase Experiment Alfred Hershey & Martha Chase worked with a bacteriophage: A virus that invades bacteria. It consists of a DNA core and a protein coat DNA Protein coat

Protein coats of bacteriophages labeled with Sulfur-35 DNA of bacteriophages labeled with Phosphorus-32 Bacterium Bacterium Phage Phage 1.Hershey and Chase mixed the radioactively-labeled viruses with the bacteria The viruses infect the bacterial cells.

Protein coats of bacteriophages labeled with Sulfur-35 DNA of bacteriophages labeled with Phosphorus-32 1.Separated the viruses from the bacteria by agitating the virus-bacteria mixture in a blender

Protein coats of bacteriophages labeled with Sulfur-35 DNA of bacteriophages labeled with Phosphorus-32 1.Centrifuged the mixture so that the bacteria would form a pellet at the bottom of the test tube 1.Measured the radioactivity in the pellet and in the liquid

Hershey & Chase’s Experiments Created labeled bacteriophages Created labeled bacteriophages Radioactive sulfur Radioactive sulfur Radioactive phosphorus Radioactive phosphorus Allowed labeled viruses to infect bacteria Allowed labeled viruses to infect bacteria Asked: Where are the radioactive labels after infection? Asked: Where are the radioactive labels after infection?

virus particle labeled with 35 S DNA (blue) being injected into bacterium 35 S remains outside cells virus particle labeled with 32 P DNA (blue) being injected into bacterium 35 P remains inside cells Fig. 13-4ab, p.209 Hershey and Chase Results

Structure of the Hereditary Material Experiments in the 1950s showed that DNA is the hereditary material Experiments in the 1950s showed that DNA is the hereditary material Scientists raced to determine the structure of DNA Scientists raced to determine the structure of DNA Watson and Crick proposed that DNA is a double helix Watson and Crick proposed that DNA is a double helix Figure 13.6 Page 211

13.2 Structure of Nucleotides in DNA Each nucleotide consists of Each nucleotide consists of Deoxyribose (5-carbon sugar) Deoxyribose (5-carbon sugar) Phosphate group Phosphate group A nitrogen-containing base A nitrogen-containing base Four bases Four bases Adenine, Guanine, Thymine, Cytosine Adenine, Guanine, Thymine, Cytosine

Nucleotide Bases phosphate group deoxyribose ADENINE (A) THYMINE (T) CYTOSINE (C) GUANINE (G)

Composition of DNA Chargaff showed: Chargaff showed: Amount of adenine relative to guanine differs among species Amount of adenine relative to guanine differs among species Amount of adenine always equals amount of thymine and amount of guanine always equals amount of cytosine Amount of adenine always equals amount of thymine and amount of guanine always equals amount of cytosine A=T and G=C

Rosalind Franklin’s Work Was an expert in X-ray crystallography Was an expert in X-ray crystallography Used this technique to examine DNA fibers Used this technique to examine DNA fibers Concluded that DNA was some sort of helix Concluded that DNA was some sort of helix

Watson-Crick Model DNA consists of two nucleotide strands DNA consists of two nucleotide strands Strands run in opposite directions Strands run in opposite directions Strands are held together by hydrogen bonds between bases Strands are held together by hydrogen bonds between bases A binds with T and C with G A binds with T and C with G Molecule is a double helix Molecule is a double helix

13.3 DNA Structure Helps Explain How It Duplicates DNA is two nucleotide strands held together by hydrogen bonds DNA is two nucleotide strands held together by hydrogen bonds Hydrogen bonds between two strands are easily broken Hydrogen bonds between two strands are easily broken Each single strand then serves as template for new strand Each single strand then serves as template for new strand

How does DNA replicate? ConservativeSemi-ConservativeDispersive Hypotheses:

Bacteria cultured in medium containing a heavy isotope of Nitrogen ( 15 N)‏Bacteria cultured in medium containing a heavy isotope of Nitrogen ( 15 N)‏ Meselson-Stahl Experiment

Bacteria transferred to a medium containing elemental Nitrogen ( 14 N)‏Bacteria transferred to a medium containing elemental Nitrogen ( 14 N)‏ Meselson-Stahl Experiment

1.DNA sample centrifuged after First replication

Meselson-Stahl Experiment 1.DNA sample centrifuged after Second replication

DNA Replication Each parent strand remains intact Each parent strand remains intact Every DNA molecule is half “old” and half “new” Every DNA molecule is half “old” and half “new” Fig. 13-7, p.212

Why the discontinuous additions? Nucleotides can only be joined to an exposed — OH group that is attached to the 3’ carbon of a growing strand. Energy for strand assembly is provided by removal of two phosphate groups from free nucleotides Fig. 13-8c, p.213 Strand Assembly

Continuous and Discontinuous Assembly Strands can only be assembled in the 5’ to 3’ direction continuous on just one parent strand. This is because DNA synthesis occurs only in the 5´ to 3´ direction. discontinuous: short, separate stretches of nucleotides are added to the template, and then ligase fill in the gaps between them.

Base Pairing during Replication Each old strand serves as the template for complementary new strand Fig. 13-8, p. 213

Enzymes in Replication Enzymes unwind the two strands - helicase Enzymes unwind the two strands - helicase DNA polymerase attaches complementary nucleotides DNA polymerase attaches complementary nucleotides DNA ligase fills in gaps (Okazaki fragments) DNA ligase fills in gaps (Okazaki fragments) Enzymes wind two strands together Enzymes wind two strands together

DNA Repair Mistakes can occur during replication Mistakes can occur during replication DNA polymerase can read correct sequence from complementary strand and, together with DNA ligase, can repair mistakes in incorrect strand DNA polymerase can read correct sequence from complementary strand and, together with DNA ligase, can repair mistakes in incorrect strand

13.4 Cloning Making a genetically identical copy of an individual Making a genetically identical copy of an individual Researchers have been creating clones for decades Researchers have been creating clones for decades These clones were created by embryo splitting These clones were created by embryo splitting

1 A microneedle2 The microneedle has emptied the sheep egg of its own nucleus. 3 DNA from a donor cell is about to be deposited in the enucleated egg. 4 An electric spark will stimulate the egg to enter mitotic cell division. the first cloned sheep Fig. 13-9, p.214Cloning

Showed that differentiated cells could be used to create clones Showed that differentiated cells could be used to create clones Sheep udder cell was combined with enucleated egg cell Sheep udder cell was combined with enucleated egg cell Dolly is genetically identical to the sheep that donated the udder cell Dolly is genetically identical to the sheep that donated the udder cell Dolly: Cloned from an Adult Cell

Fig. 13-9, p.214 Dolly: Cloned from an Adult Cell

Ian Wilmut was the first to produce a cloned sheep, which he named Dolly Ian Wilmut was the first to produce a cloned sheep, which he named Dolly Dolly experienced health problems similar to other mammals cloned from adult DNA Dolly experienced health problems similar to other mammals cloned from adult DNA Impacts, Issues: Goodbye Dolly

Fig. 13-1a, p.206 Goodbye Dolly

The risk of defects in clones is huge The risk of defects in clones is huge Possible benefit – patients in desperate need of organ transplants Possible benefit – patients in desperate need of organ transplants Genetically modified cloned animals may produce organs that human donors are less likely to reject Genetically modified cloned animals may produce organs that human donors are less likely to reject Cloning humans – ethical? Cloning humans – ethical? Impacts, Issues: Goodbye Dolly

Therapeutic Cloning SCNT – Somatic Cell Nuclear Transfer SCNT – Somatic Cell Nuclear Transfer Transplant DNA of a somatic cell from the heart, liver, muscles, or nerves into a stem cell (undifferentiated cell) Transplant DNA of a somatic cell from the heart, liver, muscles, or nerves into a stem cell (undifferentiated cell)

More Clones Cows

Fig , p.215