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

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

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

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