Section 2 Lectures 4-6

Beyond Mendelian Genetics, DNA Discovery and Replication, Genetic Code

Limitation’s of Mendel’s Ideas

* Did not explain continuous variation
* By law of independent assortment and meiosis genes must be on different chromosomes?
* Humans have 23 pairs of homologs, so do we only have 23 genes?

Sex-Linked Traits

* Traits that run in families and predominantly affect males
* Both parents must be heterozygous or it would not be visible

Red Green Colour Blindness

* Appears in 1/4 children of unaffected parents (so recessive)
* Overwhelmingly appears in males
* Only males of unaffected parents are colour blind
* Colourblind females have affected parents

What would it look like if sex and colour blindness assorted independently?

* XX Cc crossed with XY Cc

What is the actual ratio for colourblind progeny?

* The Y chromosome always has the recessive allele because it does not have a gene for colourblindness

Why does Y always have the recessive? (colourblindness)

* It is tiny compared to the X chromosome
* It does not have a gene for colourblindness
* The absence of a gene is also an allele

What are the three types of alleles?

Wild Type: The predominant allele in a population (>99%)

Mutant: A change from the wild type allele, typically the result of a recent mutation. Can refer to alleles that cause disease.

Polymorphic: An allele that is present in

What is the exception to the law of independent assortment?

* Only applies to genes on different chromosomes

What happens when someone has the colourblindness trait?

* Colourblindness gene make a protein (opsin) that detects green light. The colourblindness trait results from a gene on the X chromosome that is not functional and cannot make the protein properly. It is mutated.

Hemizygous

* When a gene is missing from one of the chromosomes

Thomas Hunt Morgan and Fly Test Cross

* Discovered linkage and surmised it was the result of 2 genes being on the same chromosome
* After crossing fruit flys, and examining their colour (black, grey) and wing type (vestigial, normal) there was anomalies in the result, so the genes do not independently assort but are linked on the same chromosome
* Anomalies because the chromatids exchange arms in prophase of meiosis 1, so if the breaking and rejoining occurs between the B and V in a BV chromatid that is exchanging with a bv chromatid, you get end up with Bv on one and bV on the other

Recombination/Recombinant Phenotypes

* Chromatids exchange arms in prophase of meiosis 1, so if the breaking and rejoining occurs between the 2 genes you get an unexpected mix (like in fruit fly test)

Genes Linked in Trans

* Alleles on different homologous chromosomes

Genes Linked in Cis

* Alleles on same homologous chromosome

Recombination Rate

* Recombination occurs at random points
* A measure of physical distance on the chromosome
* The probability/rate of recombination occurring between 2 genes depends on how far apart the two genes are on the chromosome

Locus

* Specific position on a chromosome where a particular gene or genetic marker is located

Formula for Distance Measured in centiMorgans

100( # of recombinants/ # total progeny)

Linkage Group

* Set of genes shown to be linked together on the same chromosome

Linear Order of Genes

* Genes have a linear order on the chromosome that is invariant
* Every individual of a species has the same genes linked together on the same chromosomes in the same linear order

Incompletely Dominant or Semi-dominant

* Contribution to continuous variation
* Ex. Snap Dragon flower with rr white and RR red
* Homozygous progeny are white or red
* But heterozygous progeny (Rr) turn pink
* Due to the non functional genes from the recessive allele

Summary: Three phenotypes for one gene, two alleles

Allelic Series

* Contribution to continuous variation
* More than 2 alleles per gene (in a population)
* Alleles encode gene products with varying degrees of partial function who’s effects are additive
* Ex. Alzheimer’s can be related to an allelic series

Environmental Contribution to Phenotype

* Contribution to continuous variation
* Penetrance, Expressivity, Polygenic/Multigenic, and Epistasis all tie into this

Penetrance

The % of individuals of a genotype that show the phenotype at all, sometimes you can have the same genotype and still not display the phenotype 

Expressivity

Degree to which a phenotype is expressed. As individuals with the same genotype vary in their degree of expression.

Ex. shades of pink in a flower

Polygenic/Multigenic

* Multiple genes that may contribute to a train
* Ex. at least 6 genes that influence heart attack risk

How to Purify Something

* Separate components of a complex mixture
* Assay each component to determine which has the desired property

Assay

* Way of measuring something
* Can measure a substance, like an assay for starch
* Can measure an abstract phenomenon, like an assay for memory (ex. mice in a maze)

How to Purify DNA

* grind up organism
* extract lipids and proteins with organic solvent (phenol)
* precipitate with ethanol

Friedrich Miescher isolated nuclei from pus cells…

found that the main constituent of the nucleus was a compound he called nuclein (turned out to be DNA)

Fredrick Griffith Mice Experiment

* interested in developing a vaccine for pneumonia with mice
* injected mice with live or heat killed pneumonia bugs
* either smooth (S) strain or rough (R) strain
* S would kill injected mice, R would not
* mice injected with dead S and live R would die because R transformed into S

Transforming Principle

* Substance that transformed R to S
* So S had genetic material that was heritable to R
* Can conclude transforming principle = genetic material

Aside: ability to transform R to S is an assay for genetic materialness

Oswald Avery

* identified that transforming principle was DNA

Hershey Chase Experiment

* only 2 things in a virus (DNA and protein)
* viruses attach to bacterial and insert material to get bacteria to create more virus
* wondered if material inserted was DNA or protein
* labeled protein and DNA with two different radioactive elements
* infected bacteria with virus and threw it in a blender
* caused protein coat to be sheared off of the bacteria
* put into super natant, idea was that whatever stayed back in the pellet with the bacteria was the genetic material
* now the bacteria produced more virus containing DNA marker, thus DNA must be the viral genetic material

Watson & Crick

* Tried to figure out how DNA replicates and determines phenotype, and 3D structure
* used Rosalind Franklin and Maurice Wilkin’s unpublished data

Clues to Mystery of DNA Structure

1) chemical structure of monomers

* DNA is a polymer made of 4 subunits (nucleotides AGCT)
* Polarity defined by which end of the sugar backbone was sticking out: 3’ OH or 5’ phosphate

2) Chargaff’s Rule

* showed that the amount of A = T & G = C but A+T/G+C can vary between organisms

3) Crytalography - Franklin & Wilkins

* used x-ray diffraction patterns on a photographic plate
* found DNA consists of 2 strands
* strands twist around each other in a double helix
* phosphates are likely on the outside (they are(
* strands run antiparallel to eachother

How were the structure clues used?

* came up with model where nitrogenous bases are in the middle and the phosphate backbone is on the outside
* charged phosphate backbone exposed to water
* bases are hydrophobic planar molecules, stacked on top of each other in the centre
* A always with T, G always with C
* this makes H bonds most stable
* always purine-pyrimidine pairs, keeps distance between strands constant

Reverse Complement and DNA

DNA are reverse complements of eachother.

Reverse: they are anti parallel (read in opposite directions)

Complement: A on one strand corresponds to T on the other, etc.

What does the structure of DNA suggest?

1) nucleotide sequence doesn't affect the overall structure, information is encoded arbitrarily by the sequence of base pairs

2) 2 strands encode the same information in a complementary form suggesting a method of replication

Propositions for DNA Replication Mechanisms (3)

1) Conservative Replication - 2 Strands unbounded, made a new strand and then re-annealed, and the 2 new strands re-annealed. Gives 1 old helix, 1 new helix.

2) Semiconservative Replication - Each old strand made a new strand and remained annealed to it (one that actually happens)

3) Dispersive Replication - DNA would break apart and rejoin to produce 4 strands, each with a mixture of old and new DNA

Meselson and Stahl Expiriement

* proved it was semiconservative
* labeled DNA by growing bacteria with a heaver isotope of Nitrogen
* 15N made the DNA heavy allowing it to be distinguished when centrifuged in cesium chloride
* after the 1st duplication, half as heavy, since half was normal weight
* after second duplication same thing occurred which could only occur if it was semiconservative replication

Making DNA in a Test Tube

* four nucleotides (in triphosphate nucleotide form)
* DNA polymerase - enzyme that does the polymerization reaction
* Template DNA with ragged ends so that DNA polymerase can attach the complementary nucleotide next in line
* The bond between the alpha phosphate (attached to sugar) and the beta phosphate of the nucleotide is broke
* then the alpha phosphate is attached to the 3’ hydroxyl group of the last nucleotide on the strand being extended

(DNA Synthesis 5’ → 3’)

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Note: in the test tube the DNA usually comes from an organism already purified so it has ragged ends from the damage of this process

How to pry strands apart and how to make ragged ends?

enzyme helicase: unwinds the DNA strands by expanding energy

enzyme primase: makes short complementary RNA primers that act as a ragged end so DNA polymerase can go to work

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Note: primase only acts on the lagging end

What are origins of replication?

* since helicase and primase do not randomly initiate replication, but at specific spots
* spots are called origins of replication
* bacteria have one, eukaryotes have many

Replication Forks

* helicases unwind the DNA in each direction from the origin of replication
* bubble forms with replication forks on either end where the old double stranded DNA is being split to act as the template for the formation of 2 new strands

Lagging Strand

* primase puts down short RNA stretches (primers) every few hundred bases in 3’→ 5’ direction
* DNA polymerase 3 joins these fragments (Okazaki fragments) from 5’ → 3’ direction
* DNA polymerase 1 chews up the RNA primers and uses the newly synthesized DNA as a primer to fill in the gaps
* DNA ligase joins the ends of the newly synthesized strand and replication finishes
* (joins by making phosphate bond between strands)

Circular Chromosome (DNA) Replication

* replication spreads in both directions from origin until forks moving in opposite directions meet at the opposite side of the circle
* this creates 2 linked circles that are broken and unlinked by and enzyme

Linear Chromosome Replication

* replication spreads in both directions from origin(s) until forks moving in opposite directions meet in the middle
* DNA is replicated simultaneously in parallel, at multiple origins of replication

Fixing Replication Mistakes

1) Polymerase has proofreading mechanism that immediately removes bases that are not complementary to the template strand

2) mechanism at work during recombination called mismatch repair

3) mechanism at work the rest of the time called excision repair

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Note: excision repair also works on thymine dimers formed by exposure to UV light

Language of Genes

* must be encoded in the sequence of nucleotides
* code tells the cell what proteins to make

Epistasis

* kind of inheritance
* albino and agouti genes in mice have an epistatic relationship (not additive)
* so if you are homozygous recessive for the albino gene (aa) it doesn’t matter what you are for the agouti gene (always albino)
* albino gene has “super dominance”

Why do albino and agouti genes in mice have an epistatic relationship ?

* albino gene is necessary to make a colourless chemical and make it into a coloured pigment (that ends up in fur)
* if you don’t have the albino gene you can’t make the pigment and your fur never gets coloured
* the agouti gene takes the pigment (made by albino) and distributes it in the fur
* normally it distributes it in a way that gives brown fur, but a recessive allele (bb) gives black
* the two genes interact in a way where if you are missing the ability to make pigment (recessive albino) then the agouti gene is irrelevant because there is no pigment to distribute and you will always have white fur

Alkaptonurea (Garrod)

* disease where urine is black
* if neither parent was affected the 1/4 of children was (not dominant)
* in one case an affected father has 4/8 affected children
* Garrod showed that it was a recessive hereditary trait
* deduced that it results from the absence of a specific enzyme in the metabolism

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CONCLUDED THAT GENES MAKE ENZYMES

Alkaptonurea (Beadle & Tatum)

* expanded on Garrod’s idea
* if genes make proteins (enzymes) then we should be able to find a mutant corresponding to every enzyme in a pathway

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One-gene, one-enzyme hypothesis → one-gene, one-protein

Beadle & Tatum Experiment

* used a mold, reasoned that mutants lacking enzymes necessary for a pathway would get stuck and not be able to produce the components downstream 
* would prevent mold from growing, but could be rescued by providing the missing compounds in the growth media 
* they isolated mutants that required certain metabolic precursors in order to grow (auxotrophs) as opposed to the wild type prototrophs 

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* mold needed Argenine, but not orthinine or citrilline, other than to make argenine
* ex. strain 2 could not make enzyme B (lacked gene b) so it could not turn orthinine to citrilline, but if given citrilline in its diet it could convert it with enzyme C into argenine

Central Dogma Overview

* information flow from gene to protein
* DNA is in the nucleus but proteins are manufactured in the cytoplasm by ribosomes
* RNA transfers the genetic information from the nucleus to the cytoplasm (where proteins are made)

Crick, Brenner, Jacob (Central Dogma)

* led to formulation of the central dogma
* idea that genetic information from from DNA → RNA → Protein
* proposed that the flow is unidirectional, it never goes in reverse

First Step of Central Dogma

* first step from genotype to phenotype is the formation of the messenger RNA
* RNA very similar to DNA (U instead of T), RNA has 2’ hydroxyl but that does not have an effect
* Similarity allows for information to be easily transferred
* RNA polymerase (enzyme) sits down on DNA at the beginning of a gene and unwinds
* Starts creating a complementary RNA strand, until it reaches the end of the gene

RNA Polymerase

* makes RNA like DNA polymerase but it doesn’t need a 3’ hydroxyl to get started and it incorporates triphosphate ribonucleotides
* occurs from 5’ → 3’
* new mRNA has all the info on the DNA for the gene
* mRNA leaves the nucleus and enters the cytoplasm
* ribosomes use it to make proteins

Coding Problem

Making of Amino Acids

* cannot be a 1 nucleotide per amino acid correspondence, would only make 4 amino acids (4^1)
* cannot be a 2 nucleotide per amino acid correspondence, would only make 16 amino acids (4^2)
* has to be a 3 nucleotide per amino acid correspondence, to make 64 amino acids (4^3)

Transcription

* process of making mRNA
* process where information from a DNA strand is transferred to an mRNA

First Protein that had its amino acid sequence determined

insulin

Transfer RNA Role and Structure

Transfer RNA (tRNA)

* acts as an adapter between nucleic acid and protein (float around in cell)
* at least one tRNA for each amino acid (often 1+)
* tRNA for an amino acid contains an anticodon sequence that is the reverse complement of the codon for that amino acid
* each tRNA has unique sequences that causes it to have a unique overall shape
* they all have an amino acid attachment site on the 3’ end

Experiment to figure out genetic code

* test tube has things from a cell that make proteins (ex. ribosomes)
* add a strand of RNA that you made with a repeating sequence ex. AAA, AGA
* analyze the results, which is a protein chain composed of amino acids corresponding to the RNA sequence
* ex. UUUUUUUUUUUUUU → PhePhePhe…

Reading Frames

* Gobind Khorana realized that a sequence can yield 3 different sequences
* can be read as AAG, AGA, or GAA
* now we know there is a frame for reading the code that can be shifted

Codon

* three DNA letter combination
* Stop codon: UAA, UGA, UAG, tell the ribosome the protein is done
* several different codons for the same amino acid, thus the code is said to be degenerate

Why is the code degenerate?

* DNA sequence uniquely determines the amino acid sequence of a protein
* amino acid sequence of a protein does not uniquely determine the DNA sequence

mRNA and tRNA interactions

* ribosome causes tRNA to sequentially bind to mRNA codons
* the catalyzes the polymerization of the amino acids on the other end of the tRNAs
* creates a string of amino acids, whose sequence is determined by the mRNA sequence

Translation

* process of converting the mRNA sequence to the protein sequence
* when the ribosome converts the mRNA sequence with tRNAs into a protein polypeptide 
* nucleotide sequence → amino acid sequence 

How do amino acids get attached to tRNA?

* class of enzymes, one for each amino, catalyzes the attachment of a specific amino acid to a tRNA
* enzymes = aminoacyl tRNA sunthases
* this is what the code is truly determined by

What is the code truly determined by, and how do we know?

* enzymes = aminoacyl tRNA sunthases 
* take a tRNA with phenylalanine and chemically convert to alanine
* adding modified tRNAs to a reaction mix with ribosomes, and a known mRNA, the ribosome puts an alanine everywhere there is supposed to be a Phe

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* also, can mutate the anticodon of tRNA
* ex. codon for serine to codon for Phe
* synthase will still charge the mutant tRNA with serine, but the serine will be incorporated into the protein where Phe should be

Conclusion on Origins of the Genetic Code

* genetic code is arbitrary
* codon used for each amino acid was decided by chance many eons ago and became fixed
* fixed because changing the code in any organism would generate nonfunctional proteins
* code is almost universal, so all organisms must have a common ancestor billions of years ago with the same genetic code that we use today