molecular genetics lecture

Molecular GeneticsMolecular Genetics

Paul Rindler M.S.Paul Rindler M.S.

TopicsTopics

Discovery of DNA Discovery of DNA DNA structure and replicationDNA structure and replication Gene expression and protein Gene expression and protein

synthesissynthesis Biotechnology toolkitBiotechnology toolkit VirusesViruses

Discovery of DNADiscovery of DNA

1800’s1800’s Johann Miescher isolated a phosphorous Johann Miescher isolated a phosphorous

containing acidic compound from the containing acidic compound from the nucleusnucleus

19281928 Frederick Griffith demonstrates the Frederick Griffith demonstrates the

existence of a chemical in bacteria that existence of a chemical in bacteria that caries genetic informationcaries genetic information


1930’s1930’s Various experiments identify chromosomes Various experiments identify chromosomes

as the source of genetic informationas the source of genetic information Chromosomes are composed of mainly Chromosomes are composed of mainly

proteins and deoxyribonucleic acid (DNA)proteins and deoxyribonucleic acid (DNA) The DNA molecule was considered too The DNA molecule was considered too

simple to be important so proteins were simple to be important so proteins were thought to carry the genetic informationthought to carry the genetic information


19441944 Oswald Avery showed that degradation Oswald Avery showed that degradation

of DNA and not protein resulted in loss of DNA and not protein resulted in loss of genetic informationof genetic information

Discovery of DNADiscovery of DNA Rosalind Franklin (1951)Rosalind Franklin (1951)

Generated X-ray Generated X-ray crystallography data crystallography data suggesting a double helix suggesting a double helix with phosphates on the with phosphates on the outsideoutside

Discovery of DNADiscovery of DNA Watson & Crick (1953)Watson & Crick (1953)

Used Franklin’s data to develop a model for DNAUsed Franklin’s data to develop a model for DNA Received a Nobel Prize in 1962Received a Nobel Prize in 1962

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DNA StructureDNA Structure

DNA compositionDNA composition PhosphatePhosphate SugarSugar Nucleic acids (bases)Nucleic acids (bases)

DNA StructureDNA Structure

DNA BackboneDNA Backbone Alternating Alternating

phosphates and phosphates and sugarssugars

Bases are Bases are attached to attached to sugarssugars

DNA StructureDNA Structure DNA is composed of 4 different basesDNA is composed of 4 different bases

Chargraff (1951) data suggested A-T and G-C base pairingChargraff (1951) data suggested A-T and G-C base pairing Pairs are bonded by hydrogen bondsPairs are bonded by hydrogen bonds

DNA ReplicationDNA Replication

Must be completed Must be completed prior to cell divisionprior to cell division

Helicase enzymes Helicase enzymes open the DNAopen the DNA

Polymerase Polymerase enzymes generate enzymes generate a new DNA strand a new DNA strand on each old on each old templatetemplate

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Discovery of DNA Discovery of DNA DNA structure and replicationDNA structure and replication Gene expression and protein synthesisGene expression and protein synthesis Biotechnology toolkitBiotechnology toolkit VirusesViruses

Gene ExpressionGene Expression

DNA codes for genesDNA codes for genes Genes are expressed as proteinsGenes are expressed as proteins Gene expression is regulatedGene expression is regulated The genome of every cell in your The genome of every cell in your

body is identicalbody is identical Cell type is determined by the genes Cell type is determined by the genes

the cells expressthe cells express

Protein SynthesisProtein Synthesis

DNA DNA →→ RNA RNA →→ Protein Protein TranscriptionTranscription TranslationTranslation

DNA acts as a template for RNA synthesisDNA acts as a template for RNA synthesis RNA has uracil (U) rather than thymine (T)RNA has uracil (U) rather than thymine (T)

Protein synthesis requires 3 types of RNAProtein synthesis requires 3 types of RNA Messenger (mRNA)Messenger (mRNA) Transfer (tRNA)Transfer (tRNA) Ribosomal (rRNA)Ribosomal (rRNA)


TranscriptionTranscription mRNA is transcribed from DNA mRNA is transcribed from DNA

Protein SynthesisProtein Synthesis mRNA is read by the synthesis machinerymRNA is read by the synthesis machinery 3 nucleotides make up a codon which 3 nucleotides make up a codon which

codes for 1 of 20 amino acids codes for 1 of 20 amino acids

Protein Protein SynthesisSynthesis

Transfer (tRNA)Transfer (tRNA)

Translates mRNA Translates mRNA sequence into sequence into amino acidsamino acids

80 nucleotide RNA 80 nucleotide RNA moleculemolecule

The anticodon is The anticodon is specific to 1 of 20 specific to 1 of 20 amino acidsamino acids

Protein SynthesisProtein Synthesis Ribosomal (rRNA)Ribosomal (rRNA)

2/3 RNA & 1/3 protein2/3 RNA & 1/3 protein 2 Subunits2 Subunits Coordinates protein synthesisCoordinates protein synthesis


The Genetic CodeThe Genetic Code

64 possible codons64 possible codons

1 start codon 1 start codon (AUG)(AUG)

3 stop codons 3 stop codons (UAA, UAG, UGA)(UAA, UAG, UGA)


TranslationTranslation InitiationInitiation ElongationElongation TerminationTermination


SummarySummary TranscriptionTranscription TranslationTranslation InitiationInitiation ElongationElongation TerminationTermination


MutationsMutations Base substitutionBase substitution

ConservativeConservative NonconservativeNonconservative

Insertion/deletionInsertion/deletion Frameshift Frameshift

Jumping genesJumping genes Mobile elementsMobile elements McClintock McClintock

(1940’s)(1940’s)

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Biotechnology ToolkitBiotechnology Toolkit

Isolation of genes for Isolation of genes for studystudy Recombinant DNARecombinant DNA

Gene TherapyGene Therapy Stem cell researchStem cell research CloningCloning

Recombinant DNARecombinant DNA

Isolation of genes for studyIsolation of genes for study Find a gene of interestFind a gene of interest Determine gene sequenceDetermine gene sequence Express the gene productExpress the gene product Functional analysisFunctional analysis


The ToolsThe Tools

PlasmidsPlasmids Small circular DNA moleculesSmall circular DNA molecules House sequences of interestHouse sequences of interest

BacteriaBacteria Uptake and replicate plasmidsUptake and replicate plasmids Express the gene of interestExpress the gene of interest Good bang for your buckGood bang for your buck


The MethodThe Method Insert gene of interest into a plasmidInsert gene of interest into a plasmid Bacterial transformation (often Bacterial transformation (often E.coliE.coli)) Plasmid replicationPlasmid replication Gene expressionGene expression Protein purificationProtein purification Protein analysisProtein analysis


ApplicationsApplications Determine gene functionDetermine gene function Synthesize useful proteinsSynthesize useful proteins

InsulinInsulin Pest resistancePest resistance

DNA mappingDNA mapping Gene TherapyGene Therapy

Gene TherapyGene Therapy

Stem cell researchStem cell research

Embryonic stem cellsEmbryonic stem cells Readily available from fertility banksReadily available from fertility banks Limited government resourcesLimited government resources Ethical concernsEthical concerns Compatibility problemsCompatibility problems

Adult stem cellsAdult stem cells Uses your own stem cellsUses your own stem cells Insufficient technologyInsufficient technology

Gene TherapyGene Therapy

CloningCloning

TherapeuticTherapeutic Recreate specific body partsRecreate specific body parts Too technically challengingToo technically challenging

ReproductiveReproductive Exact replica of an entire organismExact replica of an entire organism First achieved in 1997 with sheepFirst achieved in 1997 with sheep Human success in 2004 but data is Human success in 2004 but data is

questionable questionable

Gene Gene TherapyTherapy

Dolly (1997-2003)Dolly (1997-2003) Scotland 1997Scotland 1997 Nucleus removed Nucleus removed

from unfertilized eggfrom unfertilized egg Nucleus from a sheep Nucleus from a sheep

skin cell insertedskin cell inserted New egg implanted New egg implanted

into surrogate motherinto surrogate mother Died of premature old Died of premature old

ageage

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VirusesViruses

Very simpleVery simple Nonliving organismNonliving organism Uses host to Uses host to

replicatereplicate 2 life cycles2 life cycles

LyticLytic LysogenicLysogenic

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molecular genetics lecture