DNA: Genetic Material and History

• The genetic material is deoxyribonucleic acid (DNA).

Timeline of DNA

• Base pairs discovered in 1950.
• 1952: DNA crystals were photographed.
• Watson and Crick described the double-helix DNA in 1953.
• Advances in genetics occurred in 1953
• The Human Genome Project started in 2000.
• In 2003, the human genome was completed.

Historical Background

• Miescher isolated nuclei from pus (white blood cells) in 1869.
• He found a novel phosphorus-bearing substance called nuclein.
• Nuclein is mostly chromatin, a complex of DNA and chromosomal proteins.
• By the end of the 19th century, DNA and RNA were separated from proteins.
• Levene, Jacobs, et al. characterized the basic composition of DNA and RNA.

Analytical Tools

• Physical-chemical analysis often uses:
  • Ultracentrifugation to estimate the size of material
  • Electrophoresis to indicates the high charge-to-mass ratio
  • UV Absorption Spectrophotometry, the absorbance of UV light matches that of DNA
  • Elementary Chemical Analysis gives a nitrogen-to-phosphorus ratio of 1.67

Ultracentrifugation

• Protein density is less than 1.3 g/cm³.
• DNA density is approximately 1.7 g/cm³.
• RNA density is usually more than 1.8 g/cm³.

Bacterial Transformation

• Key experiments were conducted by Frederick Griffith in 1928.
• An observed change in Streptococcus pneumoniae occurred.
• Virulent (S) smooth colonies with capsules changed to avirulent (R) rough colonies without capsules.
• Heat-killed virulent colonies transformed avirulent colonies to virulent ones.

DNA as the Transforming Material

• Geneticists initially doubted the use of DNA due to its seemingly monotonous repeats of four bases.
• In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty defined the chemical nature of the transforming substance
• Techniques excluded both protein and RNA as the chemical agent of transformation.
• Treatments verified that DNA is the chemical agent of transformation of S. pneumoniae from avirulent to virulent.

Hershey & Chase Experiment

• Hershey and Chase demonstrated that bacteriophage infection comes from DNA.
• They investigated bacteriophage, a virus particle, itself a package of genes.
• Bacteriophage has no metabolic activity of its own.
• When a virus infects a host cell, the cell begins to create viral proteins.
• Viral genes replicate, and newly made genes combine with viral protein to assemble into virus particles.
• Some viruses contain DNA genes, while others have RNA genes, either double- or single-stranded.

Watson & Crick

• In 1953, Watson & Crick published the double-helical model of DNA structure.
• Chargaff had previously shown that the four bases are not present in equal proportions.

The Chemical Nature of Polynucleotides

• Biochemists determined the components of nucleotides during the 1940s.
• The component parts of DNA include:
  • Nitrogenous bases: Adenine (A), Cytosine (C), Guanine (G), and Thymine (T)
  • Phosphoric acid
  • Deoxyribose sugar

Nucleotides and Nucleosides

• RNA also has component parts.
• RNA has nitrogenous bases.
  • Uracil (U) replaces thymine
• Phosphoric acid is included.
• Ribose sugar is incorporated.
• Bases use ordinary numbers.
• Carbons in sugars are noted as primed numbers.
• Nucleotides contain phosphoric acid.
• Nucleosides lack phosphoric acid.

Purines and Pyrimidines

• Adenine and guanine are structurally related to the parent molecule purine.
• Cytosine, thymine, and uracil resemble pyrimidine.

DNA Linkage

• Nucleotides are nucleosides with a phosphate group attached through a phosphodiester bond.
• Nucleotides may contain one, two, or three phosphate groups linked in a chain.

A Trinucleotide

• The example trinucleotide has polarity.
  • The top of the molecule has a free 5'-phosphate group, referred to as the 5' end.
  • The bottom has a free 3'-hydroxyl group, referred to as the 3' end.

Summary

• DNA and RNA are chain-like molecules composed of subunits called nucleotides.
• Nucleotides contain a base linked to the 1'-position of a sugar and a phosphate group.
• Phosphate joins the sugars in a DNA or RNA chain through their 5'- and 3'-hydroxyl groups by phosphodiester bonds.

DNA Structure - The Double Helix

• Rosalind Franklin's X-ray data suggested that DNA had a helical shape.
• The data also indicated a regular, repeating structure.
• DNA was believed to require an irregular sequence
• Chargaff Base pairing rules:
  • A = T, G = C, and Purines = Pyrimidines
• Watson and Crick proposed a double helix with sugar-phosphate backbones on the outside and bases aligned to the interior.
• Structure is compared to a twisted ladder.
• Curving sides of the ladder represent the sugar-phosphate backbone.
• Ladder rungs are the base pairs.
• There are about 10 base pairs per turn.
• Arrows indicate that the two strands are antiparallel.

Summary

• The DNA molecule is a double helix, with sugar-phosphate backbones on the outside and base pairs on the inside.
• The bases pair in a specific way:
  • Adenine (A) with thymine (T)
  • Guanine (G) with cytosine (C)

Physical Chemistry of Nucleic Acids

• DNA and RNA molecules can appear in several different structural variants.

• Changes in relative humidity will cause variation in DNA molecular structure.

• The twist of the DNA molecule is normally shown to be right-handed (A-form and B-form), but left-handed DNA was identified in 1979 (Z-form).

• High humidity DNA is called the B-form.

• Lower humidity from cellular conditions to about 75% takes on the A-form.

  • Plane of base pairs in A-form is no longer perpendicular to the helical axis.
  • A-form is seen when hybridizing one DNA with one RNA strand in solution.

• When wound in a left-handed helix, DNA is termed Z-DNA.

• It's important to B-form represents an average, not a specified structure, can change locally

• If the base pairs per turn are greater it is overwound, otherwise it is underwound. Local binding can be affected by overall conformation

Variation in DNA

• The ratios of G to C and A to T are fixed in any specific organism.
• The total percentage of G + C varies over a range to 22 to 73%.
• Such differences are reflected in differences in physical properties of the DNA.

DNA Melting

• With heating, noncovalent forces holding DNA strands together weaken and break.
• When the forces break, the two strands move apart in denaturation or melting.
• Temperature at which DNA strands are 1/2 denatured is the melting temperature or Tm.
• GC content of DNA has a significant effect on Tm, with higher GC content meaning higher Tm.
• Nucleic acids absorb light at 260 nm wavelength because of the electronic structure in their bases.
• When strands come together, the close proximity quenches some of the absorbance.
• When strands separate, this quenching disappears and the absorbance rises 30-40%, causing a hyperchromic shift.
• The precipitous rise in the curve shows that the strands hold fast until the temperature.

DNA Denaturation

• In addition to heat, DNA can be denatured by:
  • Organic solvents
  • High pH
  • Low salt concentration
• GC content also affects DNA density.
  • There is a direct, linear relationship.
  • This is due to the higher molar volume of an A-T base pair than a G-C base pair.

Summary

• GC content of a natural DNA can vary from less than 25% to almost 75%.
• GC content has a strong effect on physical properties that increase linearly with GC content.
  • Melting temperature, the temperature at which the two strands are half-dissociated or denatured.
  • Density
• Low ionic strength, high pH, and organic solvents also promote DNA denaturation.

DNA Renaturation

• Two separated DNA strands can come back together under proper conditions.
• This process is called annealing or renaturation.
• the most important three factors are:
  • Temperature, which should be below the of melting temperature.
  • DNA concentration, which should be within limits so the higher the concentration, the more likely the 2 complementary with find each other
  • renaturation Time- as increase time, more annealing will occur

Hybridization

• Hybridization is a process of putting together / annealing two different nucleic acids strands
• Strands could be 1 DNA and 1 RNA
• Also could be 2 DNA with complementary or nearly complementary sequences

DNA Sizes

• DNA size is expressed in 3 different ways:
  • Number of base pairs
  • Molecular weight – 660 Daltons is the molecular weight of 1 base pair
  • Length - 33.2 Å per helical turn of 10.4 base pairs
• Measure DNA size either using electron microscopy or gel electrophoresis.

Relationship between DNA Size and Genetic Capacity

• How many genes are there in a particular piece of DNA?
  • This cannot be determined from DNA size alone, it depends on the specific content
• Factors include:
  • How much DNA is devoted to genes?
  • What is the space between genes?
• The upper limit of the number of genes a piece of DNA can hold can be estimated.

Start with Basic Sumptions

• Start with basic assumptions
  • Gene encodes protein
  • Protein is about 40,000 Da
• How many amino acids does this represent? -Average mass of an amino acid is about 110 Da -Average protein - 40,000 / 110 = 364 amino acids
  • Each amino acid = 3 DNA base pairs
  • 364 amino acids requires 1092 base pairs

Paradoxes

• C-value is the DNA content per haploid cell.
• It might be expected that more complex organisms need more genes than simple organisms.
• In general, this trend holds, there are some exceptions
• For the mouse or human compared to yeast this is correct.
• More DNA does not always mean a greater number of genes.
• It might be predicted that more complex organisms have a higher C-value due to a greater number of genes.
• This is not always the case; for example, a frog has 7 times more DNA per cell than humans.
• The observation that more complex organisms will not always have more DNA than simple organisms is called the C-value paradox.
• The most likely explanation is that DNA that does not code for genes (non-coding DNA) is present when the less complex organism has more DNA.

Summary

• There is a rough correlation between DNA content and number of genes in a cell or virus.
• This correlation breaks down in several cases of closely related organisms where the DNA content per haploid cell (C-value) varies widely.
• C*-value paradox is likely not be explained by extra genes, but by extra noncoding DNA in some organisms.