Transcript Genetics - Doc Ireland

Genetics
The Conversion of Information from
Genetics to Protein
THE DISCOVERY OF DNA AND ITS
ROLE
The Turn of the Century
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Optical Investigation
Chemical Analysis of the Nucleus
The Problem of Information
Griffith
Avery, MacCarty and McCloud
Chargaff
Watson and Crick
5’  3’ Notation
5’ – ACTG – 3’
3’ – TGAC – 5’
THE FLOW OF INFORMATION
The Flow of Information
tRNA
Amino Acids
Degradation
Replication
DNA
mRNA
Peptide
Ribosome
Transcription
rRNA
Proteins
Post Translational
Processing
rProteins
Translation
REPLICATION
Chromosomal Level
ORI
Termination Point
Molecular Level
Practical Level
• If given a replication problem you simply have to
rely on Chargaff’s Rule to fill in the missing data.
5’ – ATGGTAGCTATGTAGTAGGAATT – 3’
3’ – TACCATCGATACATCATCCTTAA – 5’
Remember that the second strand runs antiparallel
to the original strand.
TRANSCRIPTION
Operon Structure
Promoter
Gene Sequences
Terminator
• Most Gene Sequences in Bacteria are arranged
in this type of Promoter/Terminator system
• Operons (grouped gene sequences under a
single control) serve as a regulatory device.
• Most Gene Regulation occurs at the level of
Transcription.
Practical Level
• Transcription occurs in a similar fashion to
Replication, with the RNA Polymerase doing
most of the duties.
• The Hardest Part is telling the difference
between coding and template strands.
• Coding = the information
• Template = the compliment of coding that is
used to make the RNA strand
Practical Level 2
Coding
Template
5’ –CGCATGCCCGTGTGAGCGATAGT – 3’
3’ –GCGTACGGGCACACTCGCTATCA – 5’
RNA
5’ –CGCAUGCCCGUGUGAGCGAUAGU – 3’
Note: The RNA is produced from the Template,
but replicates the information on the Coding.
Also note the use of “U” in place of “T.”
TRANSLATION
Translation Mechanism
Translation Mechanism 2
The Genetic Code
Practical
5’ –CGCAUGCCCGUGUGAGCGAUAGU – 3’
5’ –CGC AUG CCC GUG UGA GCG AUA GU – 3’
MET|PRO|VAL|STOP
1. Always look for the first “Start” Codon from the
5’ end.
2. Separate the Codons
3. Translate the Codons till you reach a “Stop”
Codon
MUTATION
Mutations
• Mutations are changes to the DNA of a cell.
• Mutations occur either during Replication
or between Replication cycles.
• Mutations can be mistakes or changes
caused by external sources.
• A mutation that makes it through one
replication cycle becomes permanent.
DNA Repair and Proofing
• DNA Polymerase has a built in proofreading
function.
• The worst proofreading occurs in the viruses
while the best is in the Eukarya.
• Mistakes that make it through proofreading
are often caught by the DNA repair systems
Types of Mutations
• Structural
– Indel – Bases are inserted or deleted from the
sequence
– Substitution – Bases are changed within the sequence
• Functional
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Missense – Changes the amino acid coded
Nonsense – Changes to a STOP codon
Frame-Shift – Changes the Reading Frame
Silent – No change to amino acid coded
Practical Level
RNA 5’ – GCAUGCCAUGCAGAUGA – 3’
RNA 5’ – GC AUG CCA UGC AGA UGA – 3’
Protein
Met-Pro-Cys-Arg-STOP
RNA 5’ – GCAUGCCCUGCAGAUGA – 3’
RNA 5’ – GC AUG CCC UGC AGA UGA – 3’
Protein
Met-Pro-Cys-Arg-STOP
SUBSTITUTION / SILENT
RNA 5’ – GCAUGUCAUGCAGAUGA – 3’
RNA 5’ – GC AUG UCA UGC AGA UGA – 3’
Protein
Met-Ser-Cy-s-Arg-STOP
SUBSTITUTION / MISSENSE
Practical Level 2
RNA 5’ – GCAUGCCAUGCAGAUGA – 3’
RNA 5’ – GC AUG CCA UGC AGA UGA – 3’
Protein
Met-Pro-Cys-Arg-STOP
RNA 5’ – GCAUGCCAUGAAGAUGA – 3’
RNA 5’ – GC AUG CCA UGA AGA UGA – 3’
Protein
Met-Pro-STOP
SUBSTITUTION / NONSENSE
RNA 5’ – GC_UGCCAUGCAGAUGA – 3’
RNA 5’ – GCUGCC AUG CAG AUG A – 3’
Protein
Met – Gln – Met INDEL / FRAME-SHIFT
RECOMBINATION
Horizontal Gene Transfer
• Bacteria do not have sex, so they have evolved
a different set of mechanisms to reshuffle
genetic potential.
• The ultimate source of genetic variation is
mutation.
• Horizontal Genetic Transfer is transfer of
genetic material within a generation.
Mechanisms
• Conjugation – Exchange along a pilus
• Transduction – Exchange through defective
bacteriophage
• Transformation – The uptake of naked DNA
• Transposons – Interruption through “jumping”
elements.
BIOTECHNOLOGY
Recombinant DNA (rDNA)
• Recombinant DNA (rDNA) Technology is the
insertion of a novel genetic sequence into a
new host.
Basic rDNA Procedure
• Isolation of gene of interest and suitable
vector
• Gene of interest is inserted in vitro into vector
• Modified vector is introduced into a host
• Modified vector multiplies in host, making a
line of clones.
• These clones can be used for many purposes.
Tools of Biotechnology
• Selection – a procedure where strains with a
selective advantage of interest are favored in the
environment and therefore become more
numerous (contrast natural and artificial
selection).
• Mutation – Changes are made to selected DNA
(either directed or random) to change the
properties of the sequence.
• Often these two techniques are used in concert.
Selection Example
NA Plate
NA Plate
Select one colony and
then replica plate on two
different media
NA Plate
Plus Antibiotic
Restriction Enzymes
• Useful tools in Molecular Biology to
manipulate specific sequences of DNA
• Evolutionarily are a type of viral defense
mechanism
• Serve as the Cut function in Molecular Biology
• DNA Ligase (Replication) serves the Paste
function
Vectors
• Properties of Vectors (typically Plasmids)
– Self-replication
– Size capable of in vitro manipulation
– Protection mechanism
– Marker gene
• Cross organism manipulation can be
accomplished with shuttle vectors
• Viral Nucleic Acids can also be used
Polymerase Chain Reaction
Inserting DNA
• Various methods are used to insert foreign
DNA into host cells, typically this is the step
with the lowest efficiency.
– Transformation
– Electroporation
– Protoplast Fusion
– Ballistic Insertion
– Microinjections
Choosing the Right Host
• Which Host you use takes into consideration
many factors, here are typical hosts.
– Escherishia coli
– Bacillus spp. (such as B. subtilis or B. megaterium)
– Saccharomyces cerevisiae
– Mammalian Cells
– Plant Cells
BIOETHICS
EVOLUTIONARY THEORY
Evolutionary Theory 101
• Core Tenets of Theory
– All Populations possess Genetic Variation
– Genetic Variation is passed Generationally
– The Environment Selects the most adapted
genetic variants.
• Implication – Common descent means that
there must be a common ancestor.
LUCA
• LUCA – Last Universal Common Ancestor
• Evidence for LUCA
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Genetic Code
Conserved Information Mechanisms
Conserved Biochemical Pathways
Conserved Structural Details
• What existed before LUCA?
• Can we test the hypotheses experimentally?
Pathways Away from LUCA
G+ Bacteria
Proteobacteria
ARCHAEA
Spirochetes
Viridiplantae
Protists
Animals
Fungi
Aquifex
Bacteria
LUCA
Eukarya
Horizontal Gene Transfer and
Endosymbiosis
G+ Bacteria
Proteobacteria
ARCHAEA
Spirochetes
Viridiplantae
Protists
Animals
Fungi
Aquifex
Chloroplasts
Mitochondria
Bacteria
LUCA
Eukarya
TAXONOMY AND PHYLOGENY
Taxonomy vs. Phylogeny
• Taxonomy – A practical division of a complex
group of items based on shared
characteristics.
• Phylogeny – A division of biological groups
based on evolutionary understanding.
Classical Taxonomy
• Organisms (not viruses) are classified by the
classical Linnean System
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Domain
Kingdom
Phylum
Class
Order
Family
Genus
Species
NOT ALL MAY BE DEFINED FOR
A PARTICULAR ORGANISM
What is a Species?
• Since Prokaryotes do not have sex, it is impossible
to apply the Biological Species Concept.
• Microbiologists argue about how to define a
species.
• Functional Definition – A group of organisms that
share a common evolutionary lineage, have
conserved genetic sequences, similar biochemical
markers and a shared environmental niche.
IDENTIFICATION OF ORGANISMS
Principles of Identification
• KISS – Economically speaking identifications
should be made with the fewest number of
tests to ensure accuracy.
• All identifications now are polyphasic
• Most Identifications rely on a mix of Classical
and Genetic technologies
Classical Methodologies
• Biochemical Analysis
– Fatty Acid Profiles
– Growth Substrates
– Biochemical Byproducts
• Physical Characteristics
– Growth Characteristics
– Physical Structures
– Staining
– Serotyping
Genetic Methodologies
• RNA
– Ribotyping
– Reverse Transcriptase
• DNA
– RFLP Analysis
– DNA Sequencing
– rDNA Sequencing
– Plasmid Analysis
Dichotomous Keys
Gram Stain
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Cell Morphology
Rod
Coccus
Bacillus
A key is branching
decision pathway that
leads to a decision about
the ID of an organism
Catalase
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