DNA and Molecular Genetics

Golden Age of Genetics

• early 1900s to start of WWII
• scientists still unsure if DNA or proteins were genetic material of cell
• thought it was proteins since they had a bigger "alphabet"
• many discoveries proved that DNA in fact was the genetic material of the cell

Friedrich Meischer (1869)

• extracted DNA from fish sperm and pus of open wounds
• named it nuclein
• name changed to nucleic acid then to deoxyribonucleic acid (DNA)

Robert Feulgen (1914)

• discovered that fuschin dye stained DNA
• DNA discovered in all eukaryotic cells

P.A. Levene (1920s)

• found that DNA was made of sugar, phosphate, and 4 nitrogenous bases
• came up with idea of nucleotide monomer
• incorrectly concluded that bases' proportions were equal and that tetranucleotide was the molecule's repeating structure

start of study of genetics

• early 1900s
• link between Mendel's work and cell biologists' work led to the theory of inheritance

Garrod

• proposed the link between genes and "inborn errors of metabolism"
• question formed of what is a gene?

Frederick Griffith (1920s)

• studied difference between infectious S strain covered by capsule and non-infectious exposed R strain
• injected them into mice and ones with the S strain died
• heat-killed S strain did not kill the mice
• heat-killed S strain with R strain led to S strain in mice that killed other mice when injected into them

Transforming Factor

-found in Griffith's later experiments
-turned R strain to S strain

Oswald Avery, Colin Macleod, Maclyn McCarty (1944)

-discovered that DNA is the transforming factor in Griffith's experiment
-strong but not totally conclusive evidence
-favor for proteins as genetic material

Max Delbruck and Salvador Luria (1940s)

• bacteriophage is virus attacking bacteria
• studied one attacking E. coli
• virus injects DNA into cell, then DNA "disappears" while taking over bacteria and making new virus
• after 25 mins the host cell bursts, releasing hundreds of new bacteriophage
• phages have DNA and proteins → ideal for resolving nature of hereditary material

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Alfred D. Hershey and Martha Chase (1952)

• labeled DNA radioactive phosphorus
• 32 and protein with sulfur-32 (DNA has phosphorus and not sulfur, protein has sulfur and not phosphorus)
• radioactive S stayed on the outside but radioactive P was passed down

Erwin Chargaff (1950)

• analyzed nitrogenous bases in many organisms
• number of purines doesn't always equal the number of pyrimidines (Levene's idea)
• scientists knew DNA was genetic material but not how it did its job
• must carry information between generations, be chemically stable, relatively unchanging, and mutate (causing evolution)

Watson and Crick (1953)

• gathered data
  • Franklin and Wilkens's X-ray diffractions of crystalline DNA
  • Linus Pauling helically coiled structure
  • Chargaff's base data
• won Nobel Prize
• ball and stick model
  • originally a triple helix
  • disproven after realizing that A:T ratio was not 1:1 (Chargaff) and it required too much magnesium
  • they realized it must be a double helix with antiparallel strands
  • leading them to find possible replication mechanisms and information coded in triplets of bases

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DNA Structure

• sugar-phosphate nucleosides on sides and nitrogenous bases on the inside connected by hydrogen bonds
• complementary strands twist around each other in double helix
• A pairs with T/C pairs with G
• deoxyribose sugar

Nucleoside

• nitrogenous base + sugar

Nucleotide

• -sugar, phosphate, nitrogenous base
• -nucleic acid monomer

Pyrimidines

• cytosine, thymine, uracil
• 1 ring
• Mnemonic: King Tut and Cleopatra (with U for uracil) live in a Pyramid with 1 top
• 2 pyrimidines too small to bond in DNA

Purines

• Adenine and Guanine
• 2 rings
• 2 purines too big to bond in DNA

Conservative Replication

• somehow produces an entirely new DNA strand during replication

Semiconservative Replication

• two DNA molecules
• each had 1/2 the original DNA and an entirely new complementary strand
• existing strands were complementary templates for new strand

Dispersive Replication

• involved the breaking of the parental strands during replication
• somehow, a molecular reassembly mixing original and new fragments on each DNA strand

Meselson-Stahl

• grew E. coli on heavy (Nitrogen-15) and light (Nitrogen-14) mediums

• first generation on heavy then transferred to light

• if DNA replication is semiconservative, then DNA grown on light medium would be intermediate between heavy and light

• it was

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DNA Replication Process

• requires a lot of ATP regenerated in G2 phase
• occur once per cell generation
• 50 nucleotides/second in humans
• 500 nucleotides/second in prokaryotes
• occurs in the S phase

DNA Helicase

• unzip the helix by the nucleus
• breaking hydrogen bonds between bases
• forms replication fork at origin of replication (specific nucleotide set)

DNA Polymerase

• places new nucleotides in the replication fork

Replication Bubble

• an unwound and open region of a DNA helix where DNA replication occurs
• 1 in prokaryotes, multiple in eukaryotes
• entire DNA molecule's length is replicated as the bubbles meet DNA copying direction
• 5' to 3' (carbon 5 in first sugar down to carbon 3 in last sugar)
• reads from 3' to 5' (because strands are antiparallel)
• opposite directions on each strand (because they are antiparallel)
• polymerase has to attach to the OH (hydroxyl) group

Lagging Strand

• copied in fragments
• DNA Polymerase started near the end and has to continuously go farther back to carry the rest of the DNA

Okazaki fragments

• Small fragments of DNA produced on the lagging strand during DNA replication
• joined later by DNA ligase to form a complete strand.

DNA Ligase

• enzyme that joins the Okazaki fragments
• places nucleotide pairs in uncompleted parts to create 1 continuous strand

Leading Strand

• strand where DNA is copied continuously without breaks in the middle

Lagging Strand Polymerase

• synthesizes new lagging strand

DNA Adenine and Thymine Bonds

• compatible and opposite electrical charges
• 2 hydrogen bonds

Guanine and Cytosine Bonds

• compatible and opposite electrical charges
• 3 hydrogen bonds

DNA Supercoiling

• done by nucleosomes
• wrapped around histones
• stack into solanoids
• extended, condensed, then turned into mitotic stage
• approx 2m of DNA in 10 um cells
• packs and organizes DNA for cell division and gene expression
• when permanent, allows cell specialization
• active chromatin transcription promoted or inhibited by associated histones

DNA Polymerase III

• catalyzes phosphodiester bonds between sugars and phosphate groups
• proofreads complementary base pairings
• mistakes about once per billion
• carry nucleoside diphosphates

DNA Polymerase I

• removes the RNA primer and replaces it with DNA

RNA Primase

• puts RNA primer in gaps
• leads to DNA synthesis

RNA Primer

• short piece of RNA needed for DNA polymerase to start
• 10 base pairs of RNA nucleotides
• attachment and initiation for DNA polymerase III

DNA Gyrase

• relaxes supercoiling ahead of the replication fork
• prevents strands from rejoining
• no hydroxyl group

Dideoxynucleotides (ddNTPs)

• missing the 3' hydroxyl (OH group)
• terminate DNA replication since the DNA polymerase III cannot bond with them

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