DNA as Genetic Material: Experiments

Bacterial Genome Replication and Expression - Part 1

• Early research was crucial in discovering that genetic information is stored in DNA.
• The researchers Griffith, Hersey and Chase, Avery, Macleod and McCarthy shed light on how genetic information is carried.
• DNA is a double-stranded helix consisting of nucleotides with sugar, nitrogenous bases, and phosphate.
• RNA is mostly single-stranded with a different sugar and base composition than DNA and folds to gain its function.
• Proteins are chains of amino acids, each having a central carbon, an amino group, a carboxyl group, and different side chains.
• Proteins are made from 20 amino acids linked by peptide bonds.
• Proteins fold into structures to become functional.

Genetic Information Storage

• One of the primary questions in genetic research was identifying the molecule that stores genetic information.
• Protein is a larger and more complex molecule made of at least 20 different amino acids.
• DNA is a smaller, less complex molecule containing only 4 nucleotides.

Fred Griffith's Experiment (1928)

• Demonstrated the transfer of virulence (ability to cause disease) in Streptococcus pneumoniae.
• Mice injected with smooth S. pneumoniae (with a capsule) died because the capsule prevents phagocytosis.
• Bacteria with capsules appear as smooth colonies.
• Mice injected with non-virulent rough S. pneumoniae (lacking a capsule) did not die, indicating the rough strain is harmless.
• A capsule is essential for S. pneumoniae to survive and cause mortality in mice

Continued Findings of Fred Griffith's Experiment

• Injecting with a heat-killed smooth (virulent) strain of S. pneumoniae did not cause death.

• This suggests that the organism needs more than just a capsule to cause mortality.

• Injecting a mixture of heat-killed smooth (virulent) and live rough S. pneumoniae resulted in death.

• Heat-killed smooth organisms alone cannot kill the mice, although it has the capsule.

• Live rough organisms alone could not kill the mice because it needs the capsule to protect it from the immune system of the mice.

• The information that was transferred gave the rough cells (non-virulent) the ability to produce capsules, hence surviving the immune system.

Conclusion of Griffith’s Experiment

• Genetic information can be passed between organisms, converting a non-virulent cell into a virulent one.
• This phenomenon is termed the Griffith transformation, but the actual molecule carrying the genetic information remained unknown at the time.

Oswalt Avery, Colin Macleod, and Maclyn McCarthy (1944)

• Continued on prior research to determine the molecule responsible for converting non-virulent S. pneumoniae into a virulent strain.
• Smooth cells (virulent S cells) were used to create an extract containing DNA, RNA, and protein.
• Rough (non-virulent R cells) were mixed with the extract, and it was observed that the rough cells transformed into smooth cells (virulent).
• The extract was then divided into three tubes.

Continued Findings of Avery, Macleod, and McCarthy Experiment

• Tube 1 contained the extract and DNase, an enzyme added to destroy DNA but not RNA and protein.
• Tube 2 had the extract and RNase, which destroys RNA but not DNA and protein.
• Tube 3 contained the extract and proteinase, destroying proteins, but not DNA and RNA.
• The tubes were mixed subsequently with R cells of S. pneumoniae to check for R cells turning into S cells
• It was then that only the tube treated with DNase did not cause transformation of R cells into S cells.
• Transformation, in short, requires a transfer of genetic information, and it can't proceed without DNA.

Alfred D. Hershey and Martha Chase (1952)

• Bacteria have the ability to transfer genetic material to other bacteria.
• Hershey and Chase investigate the transfer events in viruses.
• They wanted to learn firstly, which part of a virus carries its genetic info into E. coli.
• and secondly, find out can the T2 bacteriophage, which attacks E. coli, enter the cell.

How Hershey and Chase Conducted the Experiment

• T2 DNA was made radioactive with 32P.
• T2 protein (coat) was made radioactive with 35S.
• Radioactive T2 viruses were mixed with E. coli and waited for the virus to infect E. coli in a test tube.
• The E. coli cells were then centrifuged to form a pellet (cells), and the unattached viruses were removed from the supernatant.

Conclusion of Hershey and Chase Experiment

• E. coli cells were resuspended in a buffer after infection.

• A blender was used to agitate the cells in order to detach any virus particles that were on the surface of the E. coli.

• Centrifugation was performed again to separate the E. coli cells from the buffer.

• The radioactivity of all the supernatant, and cells was tested and results came back.

• The 32P radioactivity (DNA) was outside the cells, 35S radioactivity (protein) was outside the cell.

• This means only DNA was injected into the cell.

• DNA is the carrier of genetic information for T2 bacteriophages.

DNA and RNA Structure

• Nucleic acids that is, DNA and RNA, are polymers of nucleotides.
• A nucleotide is a molecule of a nitrogenous base, sugar, and phosphate group, linked by phosphodiester bonds.
• A nucleoside is what you call a molecule with ribose (sugar) that attaches to a nitrogenous base.
• A deoxynucleoside is when deoxyribose (sugar) attaches to a nitrogenous base,

Nucleotide Composition

• Nucleotides are composed of a purine or pyrimidine nitrogenous base, a ribose or deoxyribose sugar, and a phosphate group.
• Both purines and pyrimidines are types of nitrogenous bases.

Nitrogenous Bases in DNA

• The nitrogenous bases in DNA includes, adenine, guanine, cytosine, and thymine.
• Adenine and guanine are categorized as purines.
• Thymine and cytosine are classified as pyrimidines.

Basic Differences Between DNA and RNA

• DNA and RNA are both made of nucleotide polymers.
• Nucleotides are linked together by phosphodiester bonds.
• They are NOT polymers of nucleotides.
• Their differences are, their nitrogenous bases, the type of sugar in it, and whether they are single or double stranded.

DNA Structure

• DNA nucleotides consist of a sugar is deoxyribose.
• DNA consist of Bases of are adenine, guanine, cytosine, and thymine.
• Phosphate is esterified to sugar carbon to form a sugar phosphate backbone.

Role of Phosphate in DNA

• In the DNA molecule, each phosphate molecule links DNA sugars together.
• The hydroxyl of one molecule, makes covalent bonds with another molecule by phosphodiester bonding which makes it attached.

DNA Complementary Strands

• The DNA double helix is held together by phosphodiester bonds (sugars on the outside) and hydrogen bonds (bases on the inside).
• A purine on one strand always pairs with a pyrimidine in complementary base pairing.
• Adenine (a purine) pairs with thymine (a pyrimidine) via 2 hydrogen bonds.
• Guanine (a purine) pairs with cytosine (a pyrimidine) via 3 hydrogen bonds.

Features of DNA Double Helix

• The sugars are right side up in one strand and wrong side up in the other.
• DNA backbones (sugars linked via phosphodiester bonds) are antiparallel as they run in opposite directions.
• One strand is 5' to 3', the other 3' to 5'.
• Bases on the strands bind according to base-pairing rules (A-T and G-C, in DNA) and are called complementary.
• The sugars are not directly opposite each other (they are offset), resulting in major and minor grooves in the helix.
• The DNA helix turns counterclockwise from above and contains 10.5 base pairs per turn.

RNA Structure

• RNA is a polymer of nucleotides with sugar of ribose and a phosphate group
• RNA bases are typically adenine, guanine, cytosine, and uracil.
• The phosphate is bonded to sugar to form a sugar-phosphate backbone,
• Most RNA tend to be single stranded in molecule construction.
• In other circumstances, some RNA parts coil back on themselves to form hairpins in its double strands by complimentary base paring.
• Because of it's design, RNA structures are important for their role in cell function.

Amino Acid Structures

• Amino acids each contain a:
  • Central carbon.
  • Attached carboxyl group (C-terminal)
  • Amino group (N-terminal)
  • Variable side chain
• Each acid, contains different properties like polarization and electrical charge depending on its side chain.

Protein Structures

• Amino acids are able to produce peptide bonds and linked up to produce polymers.
• The C-N bonds happen due to carboxyl and amino building up to produce a polypeptide.
• Polypeptide ends are polar with carboxyl (C terminus) at an end and amino group (N terminus)
• Amino acid chains are able to rotate around them to form, primary, secondary and tertiary arrangements.

Arrangement and Bonds In Proteins

• The protein chain has to be produced in a way that the amino bonds (peptide chains) are bound together by a straight structure to produce the Primary structure.
• Secondary structures are products amino acids turning into spiral or sheets to form their bonds.
• For these structures to work, their arrangement and bonds should bind to the chain.
• To even produce an even more stronger bond, quaternary structures can produce multiple arrangements to construct the whole structure.
• Because multiple forces are required to bind the structure, they must be arranged correctly.

Explore landmark experiments such as Griffith's transformation experiment, Avery, MacLeod, and McCarty's DNA identification, and the Hershey-Chase experiment. Understand how these studies proved DNA, not protein, is the genetic material. Learn about the use of radioactive isotopes and enzymatic treatments in these critical experiments.