Chapter 14: DNA: The Genetic Material (bio 1510) 3/23/21

A single strand of DNA extends in a_________ direction.

- 5′ to 3′

Avery's results were not widely accepted at first because many biologists continued to believe that proteins were the genetic material. But additional evidence supporting Avery's conclusion was provided in 1952 by______________, who experimented with viruses that infect ______. These viruses are called .....

- Alfred Hershey and Martha Chase -bacteria -bacteriophages, or more simply, phages.

What were Griffiths results for the mice experiment with Heat-killed virulent (S) strain cells and Heat-killed virulent (S) strain + live nonvirulent (R) strain cells?

-Heat-killed virulent (S) strain cells did not kill the mice -Infecting mice with a mixture of heat-killed S form with live R form caused pneumonia and death in the mice (Heat-killed virulent (S) strain + live nonvirulent (R) strain cells killed the mice)

The process/stages of replication:

-Initiation - replication begins -Elongation - new strands of DNA are synthesized by DNA polymerase -Termination - replication is terminated

Something to copy includes... Something to do the copying includes.. Building blocks to make copy includes...

-Parental DNA molecule -Enzymes -Nucleotide triphosphates

We refer to this pattern of base-pairing as ___________, which means that although the strands are not identical, they each can be used to specify the other by base-pairing.

-complementary

Note that this configuration also pairs a two-ringed purine with a single-ringed pyrimidine in each case, so that the diameter of each base-pair is the same and is considered _________

-consistent

Watson and Crick did not _____________________-; rather, they built detailed molecular models based on the information available.

-perform a single experiment themselves related to DNA structure

James Watson and Francis Crick(1953) deduced the ______________ using evidence from Chargaff, Franklin, and others

-structure of DNA

Three Possible Models of DNA Replication:

1. Conservative model - both strands of parental duplex remain intact and new DNA copies consist of all new molecules(The conservative model produces one entirely new molecule and conserves the old) 2.Semiconservative model - daughter strands each consist of one parental strand and one new strand (the two strands of DNA unwind from each other, and each acts as a template for synthesis of a new, complementary strand. This results in two DNA molecules with one original strand and one new strand) 3.Dispersive model - new DNA is dispersed throughout each strand of both daughter molecules after replication

Meselson and Stahl compared their experimental data with the results that would be predicted on the basis of the three models:

1. Conservative model was rejected because two densities were not observed after round one. 2. Semiconservative model was supported and consistent with all the observations: 1 band( single density) after round 1 and 2 bands(two densities) after round 2(After one round of replication, a single density would be predicted because all DNA molecules would have a light strand and a heavy strand. After two rounds of replication, half of the molecules would have two light strands, and half would have a light strand and a heavy strand—and so two densities would be observed.) 3. Dispersive model was rejected because the first round results were consistent and the second round, DNA heated before centrifugation, 2 bands observed, 1 light, 1 heavy

The three models for DNA replication were evaluated in 1958 by Matthew Meselson and Franklin Stahl. To distinguish between these models, they labeled DNA and then followed the labeled DNA through two rounds of replication. -Bacteria were grown in a medium containing ___, which became incorporated into the bases of the bacterial DNA -After several generations, the DNA of these bacteria was ... -Meselson and Stahl then transferred the bacteria from the ______________and collected the DNA at various time intervals. -In this experiment, DNA was extracted from the cells at ...

15N -denser than normal DNA. -15N medium to 14N medium -various time intervals.

A forms __ hydrogen bonds with T

2

Wrap around 1 axis of the double helix width:

2 nm

G forms __ hydrogen bonds with C

3

The chain of nucleotides has a ________ orientation. This the molecule has an ____________

5′-to-3′ -Intrinsic polarity -5′ phosphate group at one end and a free 3′ hydroxyl group

The Meselson-Stahl experiment *look at figure 14.11*

Bacteria grown in heavy 15N medium are shifted to light 14N medium and grown for two rounds of replication. Samples are taken at time points corresponding to zero, one, and two rounds of replication and centrifuged in cesium chloride to form a gradient. The actual data are shown at the bottom with the interpretation of semiconservative replication shown schematically.

The phage used by Hershey and Chase contains only DNA and protein, providing the simplest possible system to differenti- ate the roles of DNA and protein. If they could identify the molecule injected into the cell, they would identify the viruses' genetic material. To do this, they needed a method to introduce unique labels into both DNA and protein. Nucleotides contain phosphorus, but proteins do not, and some amino acids contain sulfur, but DNA does not. Bacteriophage DNA and protein was labeled with radioactive's...

Bacteriophage DNA was labeled with radioactive phosphorus (32P). Bacteriophage protein was labeled with radioactive sulfur (35S).

The bacteriophage used by Hershey and Chase contains only ....

DNA and protein

Avery, MacLeod, & McCarty's experiments supported what?

DNA as the genetic material

Conclusion of Hershey and Chase experiment:

DNA is the genetic material, and not protein

Action of DNA polymerase *look at Figure 14.12*

DNA polymerases add nucleotides to the 3′ end of a growing chain. The nucleotide added depends on the base that is in the template strand. Each new base must be complementary to the base in the template strand. With the addition of each new nucleoside triphosphate, two of its phosphates are cleaved off as pyrophosphate.

DNA polymerase

Enzyme involved in DNA replication that joins individual nucleotides to produce a DNA molecule(New DNA is made by enzymes called DNA polymerases, which require a template and a primer (starter) and synthesize DNA in the 5' to 3' direction.)

Somehow, the information specifying the polysaccharide coat had passed from the dead, virulent S bacteria to the live, coatless R bacteria in the mixture, permanently changing the coatless R bacteria into the virulent S variety (Griffiths experiment). What did Griffith call this and why? What is our modern interpretation of this?

Griffith called this transfer of virulence from one cell to another transformation(Information specifying virulence passed from the dead S strain cells into the live R strain cells (live cells were transformed phenotypically). Our modern interpretation is that genetic material was actually transferred between the cells.

Hershey-Chase experiment showed DNA is genetic material for phage. *look at Figure 14.2*

Hypothesis: DNA is the genetic material in bacteriophage. Prediction: The phage life cycle requires reprogramming the cell to make phage proteins. The information for this must be introduced into the cell during infection. Test: DNA can be specifically labeled using radioactive phosphate(32P), and protein can be specifically labeled using radioactive sulfur(35S). Phage are grown on either 35S or 32P, then used to infect cells in two experiments. The phage heads remain attached to the outside of the cell and can be removed by brief agitation in a blender. The cell suspension can be collected by centrifugation, leaving the phage heads in the supernatant. Result: When the experiment is done, only 32P makes it into the cell in any significant quantity. Conclusion: Thus, DNA must be the molecule that is used to reprogram the cell. Further Experiments: How does this experiment complement or extend the work of Avery on the identity of the transforming principle?

DNA polymerase matches..

Matches existing DNA bases with complementary nucleotides and links them

What were Griffiths results for the mice experiment with Live virulent (S) strain cells and Live nonvirulent (R) strain cells ?

Mice infected with the virulent S form died from pneumonia (Live virulent (S) strain cells killed the mice) Whereas infection with the nonvirulent R form had no effect (Live nonvirulent (R) strain cells did not kill the mice)

Avery, MacLeod, & McCarty first prepared the mixture of dead S Streptococcus and live R Streptococcus that Griffith had used. Then they removed as much of the protein as they could from their preparation, eventually achieving 99.98% purity. What did they find?

Removal of all protein from the transforming material(=cell extract from dead S cells) did not destroy its ability to transform R strain cells.

Avery, MacLeod, & McCarty repeated..

Repeated Griffith's experiment using purified cell extracts

Three possible models for DNA replication *look at Figure 14.10*

The conservative model produces one entirely new molecule and conserves the old. The semiconservative model produces two hybrid molecules of old and new strands. The dispersive model produces hybrid molecules with each strand a mixture of old and new.

Base-pairing holds strands together *look at Figure 14.9*

The hydrogen bonds that form between A and T and between G and C are shown with dashed lines. These produce A-T and G-C base- pairs that hold the two strands together. This always pairs a purine with a pyrimidine, keeping the diameter of the double helix constant.

Nucleotide subunits of DNA and RNA. *look at Figure 14.3* (You do not have to know the chemical structures of the bases; know which ones are purines vs. pyrimidines; know which ones have two rings vs. one ring)

The nucleotide subunits of DNA and RNA are composed of three components: a 5-carbon sugar (deoxyribose in DNA and ribose in RNA); a phosphate group; and a nitrogenous base (either a purine or a pyrimidine). Purines: adenine and guanine (two ringed) Pyrimidines: cytosine, thymine, uracil (single ringed)

Structure of a single strand of DNA *look at Figure 14.7*

The phosphodiester backbone is composed of alternating sugar and phosphate groups. The bases are attached to each sugar.

What did Rosalind Franklin discover

The structure of DNA is helical (twisting)

Antiparallel strands meaning of the double helix:

The two strands of DNA run in opposite directions

what did Hershey and chase want to identify/determine?

Wanted to determine which of these molecules is the viruses' genetic material that is injected into the bacteria(If they could identify the molecule injected into the cell, they would identify the viruses' genetic material.)

Rosalind Franklin's X-ray diffraction *look at Figure 14.5*

a. Rosalind Franklin. b. This X-ray diffraction photograph of DNA fibers, made in 1953 by Rosalind Franklin, was interpreted to show the helical structure of DNA. (

In the Hershey and chase experiment, only the bacteriophage DNA (as indicated by the 32P) entered the bacteria and was used to produce more ..

bacteriophage.

phosphodiester bond *look at figure 14.4 for example of this bond*

bond between adjacent nucleotides formed by a dehydration reaction

Base-pairing

bonds in DNA can form only between adenine and thymine and between guanine and cytosine -two strands were held together by formation of hydrogen bonds between bases on opposite strands. These bonds would result in specific base-pairs

In the experiment with Avery, MacLeod, & McCarty, DNA resembled the properties of this substance: DNA digesting enzymes did what?

destroyed all transforming activity(removed aspects of the cells(protein, RNA, and carbohydrates) all one at a time to test each. With everything that they took out, they got transformation except with DNA.)

what did James Watson and Francis Crick propose?

each DNA molecule is actually made up of two chains of nucleotides that are intertwined—the double helix.

There are two forms of streptococcus pneumoniae:

form that causes pneumonia, and form that does not cause pneumonia

The agent responsible for transforming Streptococcus went undiscovered until 1944. In a classic series of experiments, Oswald Avery and his coworkers Colin MacLeod and Maclyn McCarty did what?

identified the substance responsible for transformation in Griffith's experiment.

what did Griffith do to understand the difference between the strains?

infected mice with these bacteria

Although each individual hydrogen bond in base pairing is weak, thousand or millions of these bonds together make a very stable ...

molecule.

DNA is a ...

nucleic acid

Replication requires three things:

something to copy, something to do the copying, and the building blocks to make the copy.

This finding led to the question of what component of chromosomes actually contains genetic information. Specifically, biologists wondered which molecule carries genetic information. They knew that chromosomes are composed primarily of both protein and DNA. Which of these organic molecules actually makes up the genes? Frederick Griffith, was trying to make a vaccine that would protect against influenza, which was thought at the time to be caused by the bacteria ...

streptococcus pneumoniae.

The phosphodiester backbone is repeating..

sugar and phosphate units joined by phosphodiester bonds

In the Hershey and chase experiment, radioactive molecules were ..

tracked

The double helix is.. *look at figure 14.8 for an example of what it looks like*

two strands twisted around each other, like a winding staircase -2 strands arranged as a double helix

What did Rosalind Franklin do?

use X-Ray diffraction to get information about the 3-D structure of DNA

DNA is composed of nucleotides:

•5-carbon sugar called deoxyribose •Phosphate group (PO4) -Attached to 5′ carbon of sugar •Nitrogenous base -this base contains adenine, thymine, cytosine, guanine •Free hydroxyl group (—OH) -Attached at the 3′ carbon of sugar

All DNA polymerases that have been examined have several common features:

•Add new bases to 3′ end of existing strands •Synthesize in 5′-to-3′ direction •Require a primer of RNA/to begin synthesis

Chargaff observed an important underlying regularity in the ratios of the bases found in native DNA: What did Erwin Chargaff determine?

•Amount of adenine = amount of thymine. •Amount of cytosine = amount of guanine. •Always an equal proportion of purines (A and G) and pyrimidines (C and T)

what are the 2 strains of Streptococcus pneumoniae?

•S(smooth - has polysaccharide capsule) strain is virulent form •R(rough - lacks polysaccharide capsule) strain is nonvirulent form