DETERMINING THE GENETIC MATERIAL IN ORGANISMS

T. H. Morgan In 1908, Morgan was working with fruit flies Male XY chromosomes, Female XX chromosomes Certain X chromosome would give the fly red eyes Another type of X chromosome would give white eyes

T. H. Morgan He determines that chromosomes must determine an organism’s trait BUT... Chromosomes are made of both DNA and proteins, so which one determined the traits??

For a couple of decades, biologists knew chromosomes contained DNA and proteins. The prevailing opinion was that the genetic material was proteins and not DNA WHY???? Because proteins are more complex than DNA

Proteins has 4 different levels of structural organization (primary  quaternary) while DNA has 2 (primary & secondary) Proteins are made of different combinations of 20 amino acids while DNA is made of 4 nucleotides

Frederick Griffith In 1928, Griffith was working with Streptococcus pneumonia bacteria trying to find a cure for pneumonia 2 types of this bacteria: “S” Type  pathogenic (causes disease and death) “R” Type  non-pathogenic (doesn’t cause disease)

Frederick Griffith He did something WACKY! He heat killed the “S” type bacteria and put into a mouse to see what would happen... The mouse lived because the bacteria had been killed

Frederick Griffith He did something WACKY! He then mixed the heat killed “S” type with the “R” type and injected it into a mouse to see what would happen... THE MOUSE DIED!

Frederick Griffith Some of the living “R” type had been transformed into the pathogenic cells. Clearly, something from the dead pathogenic cells caused this heritable change, but he still didn’t know what it was... =The Transformation Principle

Avery, McCarty, and MacLeod In 1944, Avery and his team continued the work to help prove what biomolecule was causing this transformation in Griffith’s experiment They heat killed the “S” type of cells and extracted all other biomolecules (lipids, carbs) so all that was left was the protein and nucleic acids (DNA and RNA)

Avery, McCarty, and MacLeod They mixed this “S” type with the living “R” type again but gave it 3 different treatments Test 1: treated with Proteinase (an enzyme that destroys all proteins molecules) Test 2: treated with RNAase (an enzyme that destroys all RNA molecules) Test 3: treated with DNAase (an enzyme that destroys all DNA molecules) Then he injected all into different mice to see what would happen...

Avery, McCarty, and MacLeod

Test 1: mice die, meaning transformation occurred The “R” had been transformed in “S” Proteins could NOT be the transformation principle Test 2: mice die, meaning transformation occurred The “R” had been transformed in “S” RNA could NOT be the transformation principle Test 3: mice lived, meaning transformation DID NOT OCCUR No live “S” type, only “R” DNA must be the transformation principle

Avery, McCarty, and MacLeod This experiment showed that DNA is the genetic material that determines physical traits but people were still not certain…

Erwin Chargaff Chargaff’s discoveries helped future scientists discover the structure of the DNA molecule In 1947, no one knew what DNA looked like!

Erwin Chargaff The theory believed at the time was that DNA was made of EQUAL amounts of four bases – adenine, guanine, cytosine, and thymine = Tetranucleotide Hypothesis Believed that DNA must be too simple to carry genetic information for organisms

Erwin Chargaff Chargaff was not satisfied with this hypothesis He collected several DNA samples from many different types of organisms. Using paper chromatography, he separated the DNA molecule

Erwin Chargaff Paper Chromatography Mixed DNA with an acid that breaks the covalent chemical bonds, separating the nucleotides individually Allows the mixture to travel up the paper, traveling at different speeds (because of size) and allows them to separate

Erwin Chargaff Ultraviolet Spectrophotometry Analyzes the exact amounts of bases that were present in each DNA sample

Erwin Chargaff OrganismAdenine (A)Thymine (T)Guanine (G)Cytosine (C) Human Chicken Grasshopper Sea Urchin Wheat Yeast E. coli Streptococcus Herring

Erwin Chargaff NOT equal amounts of bases  Tetranucleotide Hypothesis was wrong But he noticed that the amount of Adenine is basically equal to the amount of Thymine And the amount of Guanine is basically equal to the amount of Cytosine A=T, Adenine must base pair with Thymine C=G, Cytosine must base pair with Guanine These are … Chargaff’s Rules

Erwin Chargaff If A = T, C = G, Then A + T + C + G = 100% Examples: If an organism is comprised of 20% thymine, what is the % of guanine? If an organism is comprised of 17% cytosine, what is the % of adenine?

Hershey and Chase In 1952, Hershey and Chase use a virus called a bacteriophage (bacteria eater) Viruses only have DNA and protein for biological molecules They reproduce by invading bacteria cells and hijack the cell to force it to make more viruses The host cell then will die and break open, releases many new viruses

Hershey and Chase Grew bacteriophages in media containing certain radioactive isotopes to label DNA and proteins They had two parts to their experiment: Part 1: Radioactive Sulfur Proteins would be radioactive, not DNA Part 2: Radioactive Phosphorus DNA would be radioactive, not proteins

Hershey and Chase Allowed each radioactively labeled bacteriophage infect E. coli (bacteria cells) and let them reproduce

Hershey and Chase Blended the mixture to make the bacteriophage detach from bacteria (genetic material is inside E. coli cell)

Hershey and Chase Put both mixtures into a centrifuge to separate the mixture by different densities E. coli cells are more dense, go to the bottom Virus is less dense, so it stays on top

Hershey and Chase Results Test 1: Radioactive Sulfur (proteins) Radioactivity was found in the liquid, not inside the E. coli cells Proteins could not be the genetic material

Hershey and Chase Results Test 2: Radioactive Phosphorus (DNA) Radioactivity was found in the cells in the bottom, not in in the liquid DNA must have transferred from the virus to the E. coli DNA must be the genetic material

Rosalind Franklin Hired to improve the X-ray crystallography (can provide information about the shape and structure of a molecule) X-ray Crystallography: a stream of X-rays is directed at a crystallized substance, some rays are diffracted, or scattered, as they encounter the atoms. The scattered X-rays then interfere with each other and produce spots of different intensities and these can be recorded on a photographic film. The resulting diffraction pattern is a unique “signature” of the molecule

Rosalind Franklin Maurice Wilkins and Rosalind Franklin made these DNA X-ray diffraction patterns She deduced that basic dimensions of DNA strands, and the phosphates were on the outside of what was probably a helical structure

James Watson and Francis Crick Franklin presented her findings a lecture in which Watson was in attendance Wilkins showed Watson and Crick the X-ray diffraction data Franklin obtained Used this, along with Chargaff’s rules to come up the double helix model

James Watson and Francis Crick Franklin died of cancer in 1958 In 1962, Watson and Crick and Wilkins won the Nobel Prize for solving the structure of DNA (couldn’t be awarded to Franklin)

Meselson and Stahl In 1958, Meselson and Stahl discovered how DNA replicates itself Three ideas where thought to be how it could work: 1. Conservative: one DNA is old (conserved) and one DNA is brand new

Meselson and Stahl 2. Semi-Conservative: each DNA is has one strand that is from the old and one strand that is new

Meselson and Stahl 3. Dispersive: parent DNA is broken into pieces causing the new DNA to be in segments of new and old

Meselson and Stahl They came up with a clever test to figure out which type of replication was correct Nucleotide of DNA have nitrogen in ever base (nitrogenous bases) Labeled the “parent” nucleotides in original DNA with a heavy nitrogen 15 N When centrifuged N parent strands 15 N/ 15 N

Meselson and Stahl They would then take the DNA that was marked with heavy 15 N and introduce it to nucleotides with a lighter 14 N. When the DNA replicated, they would have to use nucleotides with a different density They made the following prediction of what would happen...

Meselson and Stahl Conservative Model Original 1 st Round:2 nd Round: 15 N/ 15 N 14 N/ 14 N 15 N/ 15 N 14 N/ 14 N

Meselson and Stahl Semi-Conservative Model Original 1 st Round:2 nd Round: 15 N/ 15 N 14 N/ 14 N 15 N/ 14 N

Meselson and Stahl Dispersive Model Original 1 st Round:2 nd Round: 15 N/ 15 N 15 N/ 14 N

Meselson and Stahl Results: Original1 st Replication What could it not be? Conservative is out!

Meselson and Stahl Results: 2 nd Replication What could it not be? Dispersive is out! DNA must replicate semi-conservatively, meaning that each daughter DNA strand is made of an old parent strand a new strand

DNA Discovery and Structure Worksheet