2 Molecular Biology Importance of molecules and bonding Bonds: Ionic – transfer of electrons, results in charged atoms or ionsCovalent – sharing of electrons; most common in organic molecules
3 Types of covalent bonds Polar – results if one element is more “grabby” for the electrons (oxygen, nitrogen)ex – Oxygen in the H2O moleculeNonpolar – electrons are shared equally, no areas of chargeImportant in shape of molecules
4 Bonds between molecules Hydrogen bonding- “attraction” between H of one molecule and an electronegative element in another molecule
5 Van der Waal forces: is the sum of the attractive or repulsive forces between molecules
6 Organic chemistry – the chemistry of Carbon compounds Most biochemical macromolecules are polymers (units linked together)For the exam, think about what elements are found in the various macromolecules.
7 Carbohydrates Main energy source Made of monosaccharides many H and OH In water, forms rings
8 Can link together to form disaccharides or polysaccharides (starches) with the loss of a water molecule (dehydration synthesis or condensation reaction)
9 When polysaccharides are taken apart, water has to be added back in: Hydrolysis
10 Important polysaccharides These are made of glucose units. Glycogen – animal starch, stored in liver and musclesCellulose – plant starch (animal can’t digest)Amylose – plant starch
11 Don’t forget when figuring out formula for the polysaccharides to subtract the water molecules! Linking 6 glucose (C6H12O6) units:
12 Proteins Made of amino acids (20) Used for structure, enzymes, hormones,transport molecules, etc.Shape very important
13 R groups? Make each amino acid unique Can confer polarity to the proteinCan be hydrophobic or hydrophilicImportant in secondary and tertiary folding
14 Amino acids are linked by peptide bonds in a condensation (dehydration) reaction Orientationis important –Carboxylgroup joinedto amino group
15 Three levels of protein structure Primary: chain of amino acidsSecondary: Beta pleats and alpha helixdue to hydrogen bondingTertiary: interactions betweenR groupsdue to ionic attractions,polarity, disulfide bridges, etc.Quaternary: attractions between chains
23 Enzymes Protein catalysts Very specific Affected by temp, pH, competing moleculesRate can be altered by amount of substrate/enzymeUsually named by what they work on
24 Enzyme LabCatalase – breaks down hydrogen peroxide into water and oxygenUsed sulfuric acid to stop reactionTitration using KMnO4 to measure amt of H2 O2 left.Measured rate
25 The rate can be defined as the amount of product formed in a period of time. Or it can be defined as the amount of substrate used in a period of time.
26 Allosteric Interactions Another molecule can bind and cause the enzyme to change shape
27 Difference in Eukaryotic and Prokaryotic Cells Prokaryotic cells do not have membrane-bound organelles such as nuclei, ER, Golgi, etc.Their energy reactions are carried on in sections of their cell membrane.They do have ribosomes , DNA and some have cell walls.
28 Developing the eukaryotic cell Think about importance of an endomembrane system (endocytosis)and endosymbiosis.
29 Cell Organelles Nucleus – control via DNA making proteins Nucleolus – stores ribosomesER – rough – site of ribosome attachment- smooth – lipid metabolism, toxin removalLysosomes – digestive vacuolesGolgi – packages, modifies proteinsMitochondria – energy (ATP) via aerobic cell. respChloroplasts – photosynthesisCytoskeletal elements – microtubules, microfilaments,support, make up other structures (centrioles, flagella, etc.)Centrioles – cell division (animal cells), anchor spindle fibers
30 Cell MembraneMade of phospholipids and integral and peripheral proteins (act as carrier molecules, enzymes, gates etc)Cholesterol – maintains fluidityHave glycoproteins and glycolipids as surface markers (receptors, MHC’s etc)Hydrophobic on inside, hydrophilic on outside
32 Differences in cells Cell walls in plant, fungi, bacterial cells Cell wall composition varies- fungi: chitin- plants: cellulose- bacteria: peptidoglycanChloroplasts in photosynthetic cells
33 Connections between cells Gap junctions – animalsPlasmodesmata – plant cells
34 Movement of materials in and out of cells Surface area to volume ratio important in determining the movement of materialsSmaller cells better!
35 Types of transportDiffusion (facilitated uses carrier molecules/channels) – passiveOsmosis – Water movement – passiveActive Transport: against conc gradient,- uses energy and carrier molecules, also includes endocytosis and exocytosis
37 Osmolarity Direction of water flow depends on solute conc WATER ALWAYS MOVES INTO A HYPERTONIC (HYPEROSMOTIC) SITUATION!Look at solute concentration to gauge water movement.
38 Water Potential pressure potential + solute potential Equation for water potential (osmotic potential)Ψ = ΨP + Ψspressure potential + solute potential(+ or -) (always -)Ψ = 0 MPa for pure waterAs you add solute, the wp becomes more negative
39 Our lab: DiffusionUsed bags of different molarities; weighed water gainDetermined the solute potential SP of potato cellsWhere graph crossed line (no gain or loss of water) gave molar concentration- Use SP = -iCRT (to figure out solute potential; C = molar conc)
40 Cell Cycle controlled by checkpoints, CDK, cyclin
41 Mitosis Keeps chromosome no. constant, no genetic diversity 2 identical cellsStages: PMATThink about what is happening to the DNA during the stages.
43 cytokinesis Actual division of cytoplasm Forms cell plate in plant cellsCleavage furrow in animal cells
44 MeiosisPurpose: to divide chromosome number in half (diploid – haploid) and to promote diversity.Results in 4 NONIDENTICAL cells due to crossing over, different arrangement of chromosomes at Metaphase I.Meiosis I: cuts chrom no in halfMeiosis II: divides chromatids
47 Meiosis is used to make gametes Some organisms such as fungi have complete bodies made of haploid cells
48 Genetics Remember ratios. One traitF2 3:1 (Aa x Aa)Two trait – Remember each organisms has two alleles for each trait!ex: tall, green plant TtGgEach gamete gets ONE of each allele pair. Think of all possibilities.ex: TG, Tg, tG, tgF2 9:3:3:1 (AaBb x AaBb)
49 Be able to relate crosses to Mendel’s laws: Law of Segregation – alleles separate during formation of gametes
50 Law of Independent Assortment: each allele separates independently of other allele in pair (ie chromosomes in Metaphase I of meiosis)
51 Test cross (backcross): use homozygous recessive to determine the genotype of an organism expressing the dominant trait to see if it is heterozygous.ex – AA or AA, mate with aaSex-linked: REMEMBER TO USE SEX-CHROMOSOMES….NOTHING ON THE Y.Probability: use what you expect from individual crossesex: AaBb x AABbprobability of getting AABB?
52 If skips a generation anywhere, recessive Pedigrees:If skips a generation anywhere, recessiveIf more in males, may be sex-linkedIf dominant, has to appear in one parentType of inheritance?
53 Linked genes will not give expected ratios Determined by amount of crossing-over resulting in recombinations of parent-typesCan use to make chromosome maps- closer genes are, less recombinations orcross-overs
54 Other things Pleiotropy: one gene, many effects Polygenic Inheritance: many genes determining phenotype, additive effectEpistasis: one gene controlling expression of another geneIncomplete dominanceCodominance
55 Genetic diseasesMay be caused by chromosome abnormalities (number and structural)Turners 45 female XOKlinefelters 47 male XXYDown’s trisomy 21- may be caused by nondisjunction during cell divisionMay be caused by gene mutations
56 NondisjunctionFailure of chromosomesto separate normally
58 Karyotypes can discern chromosome abnormalities
59 Our lab: Fruit Flies Chi-square test used to test validity of results Formulas willbe given to youon the exam.
60 This number orlower to consideryour data fits yourprediction.
61 Importance of Free Energy Ability to do work in the cell
62 Energy Transformations Laws of thermodynamics:1st energy, 2nd entropy (confusion)ATP – energy carrier moleculesubstrate level phosphorylation –transferring a phosphate from ATP toa molecule to activate itoxidative phosphorylation –using the movement of electrons to attach a phosphate to ADP to make ATP
63 What to expect on the exam…. You need to know general outcomes, places in the cell these occur, importances, etc.Pathways will probably be given for you to interpret.
64 Photosynthesis vs Cell Respiration Photosynthesis – anabolicCellular respiration – catabolic6CO H2O C6H12O H2Ophotocell respDo not memorize steps. Diagrams are usually given on the AP exam for interpretation.
65 Cellular respiration deriving energy (ATP) from food we eat Three parts: glycolysis (in cytoplasm); Krebs Cycle (matrix of mitochondria); ETC (cristae membrane) in eukaryotes.Prokaryotes carry on these processes in specialized membranes near the cell membrane.
66 Glycolysis – Glucose to 2 Pyruvates, needs 2ATP to start, makes 4 ATP, net yield 2 ATP If aerobic: pyruvate changes to acetyl Co-A (after releasing CO2) to enter the Krebs CycleKrebs Cycle generates (per turn, 2 turns per glucose) 1 ATP, 3 NADH, 1 FADH, 2 CO2Krebs cycle generates many intermediaries used in other pathwaysNADH and FADH are electron/H carriers
68 If anaerobic (no oxygen), fermentation occurs and pyruvate is changed to - lactic acid in muscle cells- alcohol and CO2 in yeast cellsNo more ATP generated, but does recycle NADH to NAD+ a to be used in glycolysis.
76 Two parts:Light-dependent (in thylakoid membranes of the grana) – light separates electrons from chlorophyll and those are passed through a series of carriers to generate ATP and eventually picked up by NADP (P in plants)Water is split generating oxygen as a waste product.The purpose of splitting water is to supply electrons to those lost in chlorophyll!ATP and NADPH go to the Calvin Cycle (light independent part)
78 Calvin Cycle – use ATP and NADPH and CO2 to make glucose
79 Our labsUsing DPIP as an electron-acceptor (replaces NADP) in the light-dependent reaction, changes color.Cell respiration: germinating vs nongerminating pea seeds, measured oxygen uptake in respirometers
85 Rate Calculations How do you calculate rate? Change in product divided by change in time.
86 Molecular Genetics DNA vs RNA sugars (deoxyribose in DNA, ribose RNA structure (double strand DNA, single RNA) bases (DNA thymine) RNA (uracil)Base pairs3 bonds more stable
87 DNA replication – semiconservative (Meselsohn-Stahl – used N14 and N15)
88 Enzymes involved: (supposedly do not need to know for exam) helicase – unwindssingle-stranded binding proteins – keepsstrands aparttopoisomerase – allows strands to unravelRNA primase – attach RNA primersDNA polymerase – add new DNA basesLigase – joins Okasaki fragmentsChromosomes are protected by telomeres during replication.
89 Leading and lagging strands DNA polymerase moves in 3-5’ directionOne side copied in one pieceOther side in pieces called Okasaki fragmentsPieces joined by ligase
90 Notice the replication proceeds in opposite directions. DNA polymerase moves in 3-5’ direction
91 Protein Synthesis Central dogma: DNA – RNA – protein Two steps Transcription – mRNA made from DNA in nucleusTranslation – mRNA (codons) match totRNA (anticodons) with their amino acids at the ribosomesEPA sites (probably too specific for exam)Amino acids joined by peptide bonds
93 Transcription steps1) initiation – RNA polymerase attaches topromoter regions (TATA box) unzips DNA2) elongation – by RNA polymerase 5 – 33) termination –RNA processing:introns removed by snRNP’sexons stayend modification; Poly A tail, 5’ cap (from GTP)
94 Translation – same steps initiation – small ribosomal subunitattaches to mRNAtRNA carrying methionine attaches P sitenext tRNA comes into A sitecontinues, original tRNA goes to E sitestops at termination (stop codon)Energy provided by GTPIn prokaryotes, both processes occur in the cytoplasm of the cell; no RNA processing
95 What happens to the proteins that are made? Those that are made on attached ribosomes:Those that are made on free ribosomes:
96 Mutations Point – change in nucleotide - silent mutation – does not changeamino acid- missense mutation – different aminoacid- nonsense mutation - changes aa tostop codonFrame Shift – deletion, addition throws reading frame off.
97 DNA organizationDNA packaged with proteins (histones) to form chromatin in beads called nucleosomesEuchromatin – DNA loosely bound, canbe transcribedHeterochromatin – DNA tightly bound, due to methylationChromatin becomes chromosomes during cell division.
98 Viruses Consist of protein coat and nucleic acid Not considered “living”, need a host cellHave lytic and lysogenic cyclesCan be used as vectors to carry genesBacteriophages – used by Hershey and Chase to prove DNA was genetic materialRetroviruses – contain reverse transcriptase for RNA DNA
99 Unfortunately DNA from retroviruses such as HIV is not proof-read so many mutations may occur.
101 Bacterial Genetics Bacteria contain plasmids Most reproduce by binary fission (asex)Ways for genetic variationconjugation with sex pilitransduction – during lytic phase of viral infection, some bacterial/viral DNA is mixedtransformation - DNA taken up fromsurroundings
102 Conjugation can result with bacterial cells gaining R plasmids for antibiotic resistance.
105 Gene Regulation RNA polymerase Regulator – Promoter – Operator – Genes All cells in an organism have the same DNA, but not all of it is turned onIn prokaryotes, have operons that direct a particular pathwayRemember RPOGRNA polymerasebinds hereRegulator – Promoter – Operator – GenesCodes forrepressorwhich can bind to the operator
106 Lac operon – inducible lactose acts as an inducer
107 Tryp operon – repressible - produces enzymes for synthesis of tryptophan; presence of tryptophan in cell cuts it off
108 Remember!Inducible operons (lac) are off and are turned on by available substrate in the cell to code for enzymes to break down the substrateRepressible operons (tryp) are on and are turned off by the product which acts as a corepressor.
109 Epigeneticschanges in gene expression or cellular phenotype, caused by mechanisms other than changes in the underlying DNA sequence, some of which are heritable.Examples of such modifications are DNA methylation and histone modificationcan modify the activation of certain genes
110 Examples of epigenetics in DevelopmentSomatic epigenetic inheritance through epigenetic modifications, particularly through DNA methylation and chromatin remodeling, is very important in the development of multicellular eukaryotic organisms. Cells differentiate into many different types, which perform different functions, and respond differently to the environment and intercellular signalling.
111 Epigenetic changes have been observed to occur in response to environmental exposure—for example, mice given some dietary supplements have epigenetic changes affecting expression of the agouti gene, which affects their fur color, weight, and propensity to develop cancer
112 MicroRNA and RNAi’sNon-coding RNA’s that downregulate mRNAs by causing the decay of the targeted mRNAsome downregulation occurs at the level of translation into protein.
113 DNA technologyRecombinant DNA – use restriction enzymes to cut DNA and gene of interest to be insertedGel electrophoresis – sort fragments by size and charteDNA fingerprinting – people have different size fragment RFLPS
116 Complementary DNA or cDNA made from mRNA using reverse transcriptase PCR
117 Our Labs DNA electrophoresis of restriction enzyme fragments -how to plot graph and read size of fragmentsTransformation experiment with pGLO, inserting plasmid with GFP into E.coli cells.- calculate transformation efficiency
118 EvolutionDarwinian evolution – by means of natural selection based on heritable traitsRemember populations evolve, not individualsEvidences for: homologies, biogeography, fossil record, molecular evidence (DNA, proteins)
119 Evolution of Populations Microevolution – looking at changes in allele frequenciesHardy Weinberg Equilibrium says gene frequencies WILL NOT CHANGE if conditions are met:- no natural selection- random mating- large populations- no gene flow (migration, immigration)
120 You have to know how to do this! p = frequency of recessive allele (can be obtained by taking the square root of the number of recessive individuals in the population)r = frequency of dominant allele (subtract p from 1)p + q = 1
124 Speciationpopulations have to be reproductively isolated (cannot interbreed and produce fertile offspring)Allopatric – geographical isolationSympatric – reproductive barriers exist in same location
126 Reproductive barriers Pre-zygotic- different mating rituals, mismatch genitals, time of mating, etc.Post-zygotic- failure of zygote to thrive or failure of offspring or grand-offspring to survive and reproduce
128 Hybrids Can complicate the issue of determining if different species If hybrids can interbreed with either parent, probably not new speciesPolyploidy (allo and auto) lead to new species in plants
129 History of Life on Earth Hypotheses of how life aroseRNA hypothesisMetabolism first hypothesisAt some time though abiotic synthesis probably did occur- Miller, Urey experiment- protobionts, coacervates
130 EndosymbiosisImportant in explaining the origin of eukaryotic cells, particularly mitochondria and chloroplast
135 Phylogeny and systematics Phylogeny – evolutionary historySystematics – classifying and determining evolutionary relationshipsKNOW how to interpret and create cladograms. Expect lots of these!Use Bioinformatics (computer programs such as BLAST) to infer phylogeny
136 Cladogram AnalysisLook for outgroups (those that have the most differences)Those with the least differences are the closest together.
141 Use of parsimony in cladistics The set-up that involves the least amount of evolutionary changesIt is considered more likely that trait B evolved only once (right hand cladogram) rather than twice (left-hand cladogram).
142 Looking at ancestryPolyphyletic - A group that does not share a common ancestor,Paraphyletic - groups that have a common ancestry but that do not include all descendantsMonophyletic - includes the most recent common ancestor of a group of organisms, and all of its descendents