BIOL105
How Can DNA be the Genetic Material?

Outline:
A. The Problem
B. Biochemical & cytological work prior to 1900
C. Experiments of Griffith, & of Avery, MacLeod, & McCarty
D. Experiments of Hershey & Chase

A. The Problem
From our discussions and lab exercise last week, we now know that proteins (as enzymes) are the workhorses of the cell and that the three-dimensional shape of a protein is based on the linear sequence of amino acids that make up that protein. But where do proteins come from? The cells in our bodies, and in all organisms, are constantly making new proteins. What directs (codes for) the synthesis of a complex sequence of amino acids that is unique for each protein?

Actually, each of us already knows the answer. We have known it since middle school. DNA is the genetic material. DNA carries the codes needed to synthesize the various proteins in the cell. Yet, how can we prove it to ourselves? There are other alternatives. Maybe proteins code for themselves! This week (2/8 - 2/12), we will discuss some key experiments that prove DNA is the genetic material and next week we will do one of these experiments ourselves.

The next three sections will briefly describe some experiments that tried to answer the question "what is the genetic material?". The second section goes along with the paper "Transformed Bacteria". In each section, there will be questions shown in blue. Try to answer these questions as you read. They will be the basis of our discussion this week.

B. Biochemical & microscopic studies prior to 1940
In 1869, Friedrich Miescher purifies "nuclein", what we now call nucleic acids (DNA and RNA). Remember that nucleic acids are one of the four major groups of biological macromolecules (polymers). Around the same time, scientists who studied animal and plant cells using microscopes determined that the nucleus is important in heredity. By the turn of the century, it was shown that the chromosomes were the important part of the nucleus and that chromosomes contained both DNA and protein.

What part of the paragraph above is consistent with DNA as the genetic material? with protein as the genetic material?

Proteins are polymers, linear sequences of connected amino acids (monomers). By the early part of this century, it was known that there were many (20) different amino acids used to make proteins. DNA molecules are also polymers, linear sequences made of nucleotide monomers. It was discovered that there are only 4 kinds of nucleotides used to synthesize DNAs.

Until the 1940's, very few people believed that DNA was the genetic material.

What is the relationship between monomer and polymer?

Based on the number of monomers used in making proteins and the number of monomers used in making DNA, can you think of a reason why people doubted that DNA could code for proteins?

C. Experiments of Griffith (1928), continued by Avery, MacLeod, and McCarty (1944)
Griffith worked with two strains of pneumococcus (now called Streptococcus pneumoniae ), a bacterial pathogen of mice: R which form rough colonies (no carbohydrate capsule) and cannot cause disease, and S which form smooth colonies (have carbohydrate capsule) and can cause disease.

Given what we know about metabolism, what would be a simple hypothesis explaining why one strain of pneumococcus makes a carbohydrate capsule, while another related strain cannot?

Griffith's experiments (goes along with the middle column on the 5th page of the article):

- inject live S cells into mice ... mice die

- kill S cells with high heat, then inject into mice ... mice live

- inject live R cells into mice ... mice live

- kill S cells with high heat, then inject dead S cells and live R cells together into mice ... mice die! When bacteria were isolated from the dead mice, they were S type!

Based on what Griffith knew in 1928, what can you conclude from these experiments?

Avery, MacLeod, and McCarty repeated Griffith's experiments and then fractionated dead S cells in an attempt to isolate the factor that allowed R cells to become S cells, the "transforming principle".

In basic terms, what kind of experiment would you do to identify the transforming principle?

The experiments of Avery's lab:

dead S cells + live R cells spread on a dish of growth medium ... some colonies of S type cells would grow

break open dead S cells and pass cell extract through a fine filter, then add the liquid that passed through the filter + live R cells to a dish of growth medium ... some colonies of S type cells would grow

What would you conclude from the experiments of Avery's lab so far?

filtered liquid from broken dead S cells:

remove carbohydrate capsule with chemical treatment and/or an enzyme which degrades the capsule ... liquid can still transform R cells into S cells

remove proteins with enzymes that degrade proteins and/or mixing the liquid with chloroform and allowing the phases to separate (like oil and water) ... liquid can still transform R cells into S cells

add alcohol to liquid in approximately 1:1 ratio and a white precipitate forms ... the remaining liquid cannot transform R cells into S cells

take the precipitate, wash it with fresh alcohol, then dissolve it in fresh buffer ... solution can transform R cells into S cells

treat solution with enzymes that degrade proteins ... solution can still transform R cells into S cells

treat solution with enzymes that degrade RNA ... solution can still transform R cells into S cells

treat solution with enzymes that degrade DNA ... solution cannot transform R cells into S cells

Why do you conclude now?

D. Experiments of Hershey & Chase
Amazingly, not everyone was convinced by the experiments of Avery and his coworkers. For those folks, it was the experiments of Hershey and Chase (1952) that finally convinced them that DNA was the genetic material and not protein. Hershey and Chase worked with a virus that attack bacteria. They knew that when viruses attach themselves to a bacterial cell (same is true for viruses that attack our cells), part of each virus is injected into the cell and the rest remains on the outside. How didthey know this?

mix virus and bacteria and incubate for several minutes

put mixture into a kitchen blender and shear any virus particles off of the outside of the bacterial cells (what speed would you use? puree?)

filter bacteria out of solution, then resuspend them in fresh growth medium

hours later, the bacteria break open and new virus particles emerge

From this data what would you conclude?

A portion of the virus that is injected into the cell must include the genetic material because the cell's metabolism is altered such that a lot of viral proteins and nucleic acids are made, and new virus particles are assembled within a few hours.

Hershey and Chase figured out ways to specifically label either the viral proteins using a radioactively-tagged amino acid or the viral DNA using a radioactively-tagged nucleotide. They wanted to know which of these molecules was injected into the bacteria cell.

Experiment 1:

mix virus containing labelled protein and bacteria and incubate for several minutes

put mixture into a kitchen blender and shear any virus particles off of the outside of the bacterial cells

filter cells out of solution

look for labelled protein ... little radioactivity inside the cells, a lot of radioactivity in the solution

What would you conclude from experiment 1?

Experiment 2:

mix virus containing labelled DNA and bacteria and incubate for several minutes

put mixture into a kitchen blender and shear any virus particles off of the outside of the bacterial cells

filter cells out of solution

look for labelled DNA ... a lot of radioactivity inside the cells, very little radioactivity in the solution

What do you conclude from Experiment 2?

Use the questions in blue to prepare for Monday's class. If you don't understand something in the reading, write it down and make sure we talk about it during class.