The Central Dogma of Genetics

Genomes & Genome evolution1Genomes and Genome Evolution - BIOL 4301/6301What to expect and some suggestions.I like think of myself as fair but reasonably toughI want people to do well but Im not willing to compromise on the material or ethical guidelines to make it happen.There is no extra credit. This is non-negotiable. Study for the exams and do well on them. Ask questions IN CLASSMakes things more interesting for meOthers probably have the same questionYoure paying, get your moneys worthInteractions with other humans tends to wake people upOffice hours!!!!!!!!!! I have them. Take advantage.I am an evolutionary biologist. This class is taught from an evolutionary perspective.

Absorb and critique anything related to the subject. This includes but is not restricted to:Popular news articles, TV shows (CSI, Bones, etc.), textbooks, wikipedia, etc. Genomics is everywhere.Bring in what you find for discussion.Website - http://davidraylab.comUsername & passwordAgain, ask questions during classAsk questions DURING CLASSDid I mention that you should ask questions during class?You WILL see pictures of my adorable children. This is also non-negotiable.

Genomes and Genome Evolution - BIOL 4301/6301What to expect and some suggestions.

Objectives: By the end of this course you should be able todescribe the methods and principle of modern genome analysis describe the components and structure of viral, prokaryotic and eukaryotic genomesexplain the basic techniques of genome sequencing and analysis describe the way genomes change over time apply principles of genomics to modern biological questions intelligently discuss the outcomes of a variety of genome projects

Course Objectives and AssumptionsAssumptions: I am assuming that youhave a working knowledge of Mendelian geneticshave a working knowledge of DNA, RNA and proteinsunderstand the basic differences between eukaryotes and prokaryoteshave a basic understanding of the concept of a genehave a working knowledge of the central dogma of Biologygive a rats behind about learning this stuffHave considered enrolling in Bioinformatics. While not required, it would be a good idea to take Caleb Phillips course

Course Objectives and AssumptionsFundamental ConceptsUNIT 17The biggest failure of science education isMost people cant discriminate between what is scientific and what is not scientific.This is due, in part, to the fact that definitions of science tend to be fairly nebulous.Moreover, any moron can get a Ph.D. 8Science A method for discovering how the world around us works Assumes that all things can be explained by natural processes Does not allow supernatural explanations Why? Rooted in hypothesis formation, observation, testing, and constant re-examination of evidence Hypotheses MUST be abandoned if they are not supported by evidence The scientific community is intensely critical of its own ideas and the ideas of others. The advantage of this isnt that mistakes arent made, its that this method pretty much guarantees that mistakes are caught quickly.

9Science Step 1. Propose as many ideas as you can think of to explain a phenomenon then pick one or several. Step 2. Try to disprove it/them. Step 3. Allow others to try and disprove it/them. Basic philosophy - Ideas that survive this process are more likely to reflect the real world than ideas that dont.

10Ways of thinkingOf these ways of thinking, science is the new kid on the blockScience is a relatively new invention (arguably only a few hundred years old, if that)But think of all the progress thats been made in those few hundred years because of scientific thinking

11Potential topics for grad student lecturesData sharing

Genomics, public health, and public policy

Genomics and population genetics

Genomics and disease gene identification

Genomics and phylogeny inference

Genome browsers

Pharmacogenomics

Metagenomics and microbiomesFundamental Biological ConceptsUNIT 113GenomeDefinition depends upon organism, organelle, or virus one is talking aboutGeneric definition: Minimum DNA complement that define an organism/organelle/virusOrganelles are not, in and of themselves, living creatures. Thus something can have a genome and not be alive.Viruses may or may not be alive, depending upon how one defines lifeThe dead have genomes too.

14Things with genomesProkaryotesMonera (bacteria)ArchaeaMitochondriaChloroplastsVirusesEukaryotesAnimalsPlantsFungiProtists

Things without genomesDirtRocksWaterAirFireBut even these things may be contaminated with genomic DNA (well, maybe not fire)

15What genomes can and cant doA genome constrains but does not dictate the features of an organismEnvironmental impactsToxins, exercise, exposure to diseaseEpigenetic impactsIf someone were to clone you?

16GenomicsThe study of genomesThe discipline that applies recombinant DNA, sequencing methods, and bioinformatics to sequence, assembly and analyze the function and structure of genomes.

17The Central DogmaDNA RNA ProteinGenerally unidirectional18Nucleic AcidsRibonucleic acid (RNA) and deoxyribonucleic acid (DNA)Composed of chains of nucleotides (ribonucleotides for RNA, deoxyribonucleotides for DNA)

19Deoxyribonucleic acidA polymer of nucleotides linked by phosphodiester bonds

Nucleic AcidsPurine vs. pyrimidineCarbon positions

Nucleic AcidsDeoxyribonucleic acidAntiparallel strands held together by hydrogen bondsStrands are complementary

Nucleic AcidsDNA in 3DPretty uncanny resemblance, dont you think?Scanning-tunneling electron micrograph

23Deoxyribonucleic acid can denature, renature & hybridizeDenaturation separation of the double helix by the addition of heat or chemicalsRenaturation the reformation of double stranded DNA from denatured DNAThe rate at which a particular sequence will reassociate is proportional to the number of times it is found in the genomeGiven enough time, nearly all of the DNA in a heat denatured DNA sample will renature.

Nucleic AcidsRibonucleic acidRibose vs. deoxyriboseThymine = 5 methyl-uracilUsually single stranded

Nucleic AcidsIntramolecular base-pairingEnhanced base-pairing capacity due to G:U bondingHairpins BulgesLoopsStem-loop structuresPseudoknots

Nucleic AcidsComplex tertiary structuresMuch more flexible than DNACapable of triple bonds and base-backbone interactionsOften molded by proteins and snoRNPsLeads to complex 3 structures with catalytic capability - ribozymes

Nucleic AcidsPCOOHONBOHPCONBOOHPCONBDNARNAOHOHOHOOHPCONBOH

Nucleic AcidsRNA WorldRNAs can have complex 3D structuresThey can store genetic informationSome RNAs known as ribozymes can catalyze reactionsThus it has been hypothesized that life may have arisen first through RNA with protein and DNA being integrated later

ReplicationDNA is replicated in a semi-conservative fashion, i.e., each daughter molecule is composed of one strand of the original molecule and one newly synthesized strand.DNA polymerase is the enzyme that catalyzes synthesis of new strands out of dNTPs.30Replication: Key pointsDNA polymerase cannot generate a new strand without a 3 OH on which to add a nucleotide. Primers are required.New strands generated from 5 to 3.Replication is bidirectional. Replication forks proceed from an initiation site in both directions.Multiple sites of initiation are found along a chromosome. Initiation sites are often AT rich as AT base pairs are less stable and thus come apart more easily.Okazaki fragments are generated along lagging strand.http://www.johnkyrk.com/DNAreplication.htmlhttp://www.dnalc.org/resources/3d/04-mechanism-of-replication-advanced.html

31RNANormally single-strandedGenerated from NTPs by RNA polymerase using DNA as a template (transcription)As with DNA replication, new strand assembled in 5 to 3 direction by phosphodiester bond formationRNA is inherently less stable than DNA32Major types of RNAMessenger RNA (mRNA) carries genetic instructions (coded in DNA) from the nucleus into the cytoplasm. mRNA molecules are often called transcripts. Ribosomal RNA (rRNA) a structural component of ribosomes (the complexes that are involved in assembling proteins based upon information in mRNA templates)Transfer RNA (tRNA) acts as carrier of amino acids during protein assemblyRegulatory RNAs Many groups; miRNAs, siRNAs, CRISPR RNAs, antisense RNAs, long non-coding RNAs33TranscriptionGeneration of an RNA strand from a DNA templateMuch of the control over cell development comes at the transcriptional level All somatic cells have same DNA but can differ tremendously in morphology and functionDifferential gene expression34Transcription: Key pointsTranscription starts at the promoter, a site along the DNA molecule where RNA polymerase binds.RNA polymerase is recruited to the promoter by transcription factors. New strand generated from 5 to 3.Only one of the two DNA strands serves as a template (antisense strand). The other strand (sense strand) has the same sequence as the mRNA molecule except dTMPs have been substituted with UMPs. Which stand is used as a template differs between genes.After transcription, mRNA undergoes post-transcriptional modifications. Generally, a methyl-guanosine cap is added to the 5 end and a tail of adenosine nucleotides (poly-A tail) is added to the 3 end.In eukaryotes, the mRNA undergoes post-transcriptional splicing introns are removed and exons are spliced together.

35Transcription modelshttp://www.johnkyrk.com/DNAtranscription.html http://www.dnalc.org/resources/3d/13-transcription-advanced.htmlA few definitionsPrecursor mRNA (pre-mRNA) or heterogeneous nuclear RNA (hnRNA): mRNA immediately after transcription and before post-transcriptional modificationMature mRNA (or simply mRNA): Transcript after post-transcriptional modifications.cDNA (complementary DNA): A DNA molecule generated in a reaction catalyzed by reverse transcriptase using mature mRNA as the template.

37rRNAAssociated with proteins to form ribosomesSeveral different rRNAsGenes that code for rRNA are typically referred to as rDNA sequencesrDNA sequences found in more or less tandem repeats in genome

38tRNAtRNA molecules deliver amino acids to ribosomes during protein synthesis (translation)tRNAs have considerable secondary structure due to base pairingClover leaf 2D structureL-shaped 3D structureThere are more than 20 tRNAs (i.e., there is some redundancy)tRNA structure is highly conserved (e.g., human tRNAs can function in yeast)http://www.myweb.ttu.edu/daray/Genomes/ribosome/ribosome/ribosome_jmol_play.html

39Amino acidsProteins are made of chains of amino acidsThere are 20 amino acids utilized by biological systemsEach codon in mRNA represents an amino acid or a start/stop signalAmino acids can be acidic (net negative charge), basic (positive charge), uncharged polar (ends have different net charges), and non-polar.Uncharged polar, acidic, and basic amino acids tend to be hydrophilic and thus are often found on the outside of proteins.Non-polar amino acids tend to be hydrophobic and thus are clustered in the middle of proteins.

40Genetic code

41Formation of a peptide bondAt physiological pH (7.0), both the amino and carboxyl groups are ionized. The peptidyl transferase ribozyme catalyzes the formation of peptide bonds with the concomitant release of a water molecule.

42TranslationConstruction of an amino acid chain (protein) by a ribosome based upon the nucleotide sequence of a mRNA moleculeWhile there are minor differences between eukaryotic and prokaryotic translation processes, most steps in translation are well conserved.

http://www.johnkyrk.com/DNAtranslation.html43Spatial separation of transcription and translation is seen in eukaryotes, not prokaryotes

44What is a gene?The structure of a typical coding gene

What is a gene? How do we identify a gene?A priori methods recognize sequence patterns within expressed genes and the regions flanking themDistinctive patterns of codon statistics (most obviously, a reduced frequency of stop codons)Proximity of start codon and known promoter sitesGT/AG pairs in exonsCodon usage statistics can be typical of genes in an organismUse a set of known genes to identify regions with similar codon usage statsBeen there, seen that methods Recognize regions corresponding to previously characterized genes.

The changing definition of a gene

46Genes vs. alleles vs. lociGene: Region of DNA that controls a discrete hereditary characteristic, often (but not always) corresponding to a single protein or RNA. This definition includes the entire functional unit, encompassing coding DNA sequences, non-coding regulatory DNA sequences, and introns.Allele: One of a set of alternative forms of a gene.Locus: The position of a gene on a chromosome. Different alleles of the same gene all occupy the same locus.Definitions from Alberts et al. (1994)47RecombinationProtein-mediated (1) exchange of a DNA region between two different DNA molecules OR (2) replacement of a DNA region in one molecule by DNA from anotherAlmost always requires at least some homology between sequences involved

48Non-homologous recombinationDuplication/deletion

RecombinationGene ConversionNon-crossover recombination replacement of one allele with an alternativeFunction and impactsRegulation of gene expressionHomogenization of genome sequence21-hydroxylase 95% of pathogenic mutations arise by gene conversion of neighboring pseudogene

RecombinationProtein-DNA interactionProtein-nucleic acid bindingMost proteins to be discussed bind specific DNA/RNA sequencesMost commonly via -helix insertion into major groove(s)H donor/acceptors+ charge on helix interaction with phosphate backboneHelix-turn-helix motif1st DNA binding domain identifiedExample in repressor animation on website-sheets may also be usedTBP example

Transcriptional regulationMost regulation takes place at the transcription levelSimple in prokaryotes - Repressors, activators, the lac operon

Transcriptional regulationMost regulation takes place at the transcription levelMuch more complex in eukaryotes

Transcriptional regulationMost regulation takes place at the transcription levelIncreased complexity in eukaryotes - -globin regulation

Gene silencingImprinting selective expression of one parental alleleNeighboring genes, Igf2 and H19, are on and off depending on parental sourceIgf2 Insulin-like growth factor 2 Highly active during fetal developmentH19 a non-coding RNAMay act as a tumor suppressorWhat is involved in this regulation?Downstream enhancerCTCF regulatory proteinICR imprinting control region

Transcriptional regulationGene silencingActivators bound to enhancer could potentially activate both genesMaternal chromosome is unmethylated in this regionLack of methylation allows binding of CTCF to ICRCTCF blocks activation of Igf2 allows activation of H19Paternal chromosome is methylated in this regionMethylation blocks binding of ICR blocks activation of H19 via MeCP2

Transcriptional regulationGene silencingBeckwith-Wiedemann syndrome (BWS)~1/15,000 birthsIncreased risk of cancer (Wilms tumor)HemihypertrophyImproper imprintingBiallelic expression of Igf2No expression of H19

Transcriptional regulationRNA interferenceLigand binding to Shine-DelgarnoRNA lifespanAlternative splicingtRNA availability/codon usage

Translational regulation

Supplemental reviewReview material to brush up on these subjects is available on the course websiteStructural tutorialsWalkthroughs of DNA synthesis, DNA replication, Transcription, Translation, Recombination, etc.