BSC 315 Midterm: Study Guide Test 3

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Biological Sciences
BSC 315
Edwin Stephenson

Study Guide Test 3 Lecture 13 DNA: transitions, transversions, insertions, deletions  Point mutations are small changes in 1 or a few base pairs o Substitutions: one base replaces another  Transition: purine changes to purine and pyrimidine to pyrimidine  Transversion: purine to pyrimidine, and pyrimidine to purine o Insertions and deletions: one or a few bases are inserted or deleted Proteins: Missense, nonsense, silent, frameshift  Missense mutation: 1 base change in a protein coding region  1 amino acid change  Nonsense mutation: 1 base change  stop codon, prematurely terminating the protein  Silent mutations: cause no change in protein sequence o Alternative wildtype allele  Frameshift mutation: translation reading frame problem o Result: inappropriate reading frame “downstream” from deletion multiple missense substitutions o All frameshifts eventually introduce a nonsense codon, often after one or more missense codons Gene expression.  Mutations in non-coding regions may affect expression  Promoter mutations: if the change prevents RNA polymerase from recognizing the promoter, transcription will not occur = no protein = severe mutation  Some mutations in the 5’UTR may affect translation  If there is a mutation in the Shine-Dalgarno sequence the ribosome doesn’t bind  no translation  no protein  severe mutations  Mutations in splice junction sequences prevent splicing. Protein includes inappropriate extra amino acids, and will include a nonsense mutation in long introns Phenotype: loss of function, gain of function.  Loss of function: allele that produces a product with reduced function o Complete loss of function: Null or amorphic o Partial loss of function: hypomorphic o Some phenotypes vary with enzyme dosage = incomplete dominance  Gain of function alleles: mutation causes extra or inappropriate activity. Almost always dominant o Hypermorphic: too much activity. Most often in negative cellular regulators. Mutation results in the failure to turn off o Neomorphic: mutation results in a new function, or function in an inappropriate location or stage o Antimorphic: mutant protein poisons normal proteins Topic 14 A Different types of bacterial phenotypes. 1. Colony Morphology a. Bacteria and unicellular eukaryotes are grown in liquid culture, or on agar (semi- solid) plates. b. Example: rough and smooth 2. Anabolic metabolism a. Simple compounds + energy  complex compounds 3. Catabolic metabolism a. Compounds from environment  CO2 + H2O + energy Detecting and analyzing auxotrophic phenotypes.  Auxotrophic mutants: cannot make an essential compound (defect in an anabolic pathway)  Auxotrophic mutants are studied by testing growth on defined media  If media contains necessary nutrients, 1 cell grows into a visible colony in 1-2 days  Identifying auxotrophic mutant o Defined media = minimal media contains salts and an organic compound as energy source Detecting and analyzing nutritional mutant phenotypes.  Catabolic mutants o Cannot break down or use particular compound as sole energy source. Defect in catabolic (breaking down) pathways  Mutant identification: o Test ability to grow on minimal medium with target compound as sole energy source. Glucose is control energy source o Most colonies are able to use lactose as sole energy source o Lac- mutant grows when glucose is present but not when lactose is the only energy source  Antibiotic Resistance o Antibiotics = bacterial poisons. Kill wildtype cells. Examples:  Penicillin/amoxicillin/ampicillin  Tetracycline  Ciprofloxacin o Antibiotic resistance:  Mutation in target protein so that antibiotic no longer binds  Enzyme with altered specificity breaks down drug  Transporter/channel proteins with altered specificity reduce intracellular accumulation  Conditional Mutations o Mutations in essential genes; cells that lack gene function die. Example:  Mutations in RNA polymerase, DNA polymerase, glycolysis enzymes, ribosomal proteins o Since bacteria are haploid (1 copy of each gene), mutations must be conditional: produce phenotype under some conditions but not others  Temperature sensitivity (most common) Topic 14 B Lactose metabolism and the logic of lac operon expression  Lactose is a disaccharide and is used as an energy source to: o Import into cell o Breakdown into monosaccharides o Lactose catabolism only occurs when glucose is absent  LacY- and LacZ- mutants do not grow on minimal medium with lactose as sole carbon source, i.e., cannot break down lactose. These mutants grow if glucose is present  LacY+ encodes permease, a lactose transporter (transports lactose into the cell)  LacZ+ encodes -galactosidase; breaks down lactose into glucose and galactose (each into glycolysis)  Logic of Lac Operon o If glucose is available, the lac genes are OFF (glucose is used in preference to lactose) o If glucose is not available is lactose available? If yes, lac genes are ON (lac genes are expressed in the absence of glucose and presence of lactose) o If glucose is not available is lactose available? If no, Lac genes OFF (enzymes for lactose catabolism are not necessary if lactose is absent) Structure and expression of prokaryotic operons  Operon: cluster of genes expressed as a single mRNA, and the regulatory sequences for their expression (genes usually have similar functions)  The lac mRNA is the transcript of 3 genes, each with its own Shine-Dalgarno sequence, AUG initiation codon and translation termination codon  Operon also contains: o CRP-binding site o P (promoter) o O (operator)  CRP-binding site, promoter and operator are cis-acting sequences The functions of trans-acting factors and cis regulatory sequences  Operator overlaps with the promoter/RNA polymerase binding site. Repressor binding to the operator physically blocks RNA polymerase from the promoter  The negative trains-acting factor (lac repressor) is a dimer, each copy of which recognizes the same DNA sequence o Fits into the major groove of DNA Positive and negative regulation  Binding of trans-acting factors is sometimes required for transcription = positive regulation. Transcription occurs only when these are bound to cis-acting sequences o Positive regulatory proteins = activators  Binding of trans-acting factors may sometimes prevent transcription = negative regulation. Transcription is blocked when these are bound to cis-acting sequences o Negative regulatory proteins = repressors  Positive regulation: a bound regulatory protein is necessary for transcription  Negative regulation: a bound regulatory protein prevents transcription even in the presence of a positive regulatory protein  Regulation of the lac operon is both positive and negative  A positive regulatory protein MUST BE bound to the CRP-binding site, and a negative regulatory protein MUST NOT BE bound to the operator  Transcription is activated when an activator binds to regulatory DNA sequences = positive regulation o The activator: complex of the activator protein CRP and cyclic AMP  Binding of CRP to DNA regulatory region is controlled by cAMP availability o CRP-cAMP complex binds to DNA and acts as an activator o CRP without cAMP does not bind to DNA  cAMP concentration varies inversely with glucose concentration o High [glucose]  low [cAMP] o Low [glucose]  high [cAMP] o Low glucose  CRP-cAMP o High glucose  CRP only  Positive: a bound protein (CRP-cAMP) is required for transcription in the absence of glucose  Negative: a bound protein (lac repressor) represses transcription in the absence of lactose o Absence of lac repressor allows transcription in the presence of lactose  Normal pattern of lac gene expression is known as “inducible” o Off when lactose is absent o On when lactose is present o Occurs via negative regulation  Some mutations cause constitutive expression o Constitutive = ON all of the time, whether lactose is absent or present  LacI+ gene encodes the lac repressor protein o LacI: transcribed independently and at all times  LacO+: the operator = DNA regulatory sequence to which lac repressor protein binds o When lactose is absent the repressor protein binds to the operator and prevents transcription o When lactose is present the repressor is inactive and does not repress transcription  Result: transcription occurs o Lactose inactivates the repressor by binding to its lactose-binding site which induces a conformational change in the protein so that it no longer binds to the operator  Lactose induces by inactivating the repressor  Why do lacI and lacO mutations have constitutive phenotypes? o The lacI+ and lacO+ functions are necessary to turn off lac operon expression  lacI-: mutant repressor protein cannot bind to operator  lacO-: mutant operator DNA sequence cannot be recognized by repressor protein Analysis of mutants in haploid and partial diploid cells led to an understanding of regulatory mechanisms.  Since lacI- and lacO- have the same phenotype, how was the cis-trans model for regulation discovered? o [Cis-trans: repressor protein (trans) binds to operator (cis)]  Plasmids: small circular “optional” chromosomes o Plasmid that determines mating type (F factor) o Plasmids with antibiotic resistance genes  F factor may integrate into chromosome  Imprecise “excision” carries some chromosome genes on the F factor (now called F’ factor) o Example: integration near the lac operon o F’ factor contains the lac operon (F’ lac) o Transfer of F’ lac to a normal strain by mating creates a strain with 2 copies of lac operon = merodiploid Topic 15 Chromatin structure and effects on gene expression  Eukaryotic nuclear DNA is packaged with protein o Combination of DNA + protein = chromatin  Protein: histones and non-histone proteins o Histones  5 protein types. Small (~100 amino acids), basic (high content of arginine and lysine) o Non-histone proteins  Thousands of types. RNA polymerase, splicing factors, regulatory proteins  Nucleosome: fundamental unit of chromatin. Structure: o 8 core histone proteins, 2 of H2A, H2B, H3 and H4 o ~160 base pairs of DNA wraps around the outside th  5 histone, H1, associates with DNA entering and leaving  `40 base pairs of DNA between nucelosomes (= linker DNA)  DNA packaging: o All chromosomes: 2 meters of DNA/cell  nucleus of 6 M diameter o Average single chromosome: 4cm DNA molecule  10 M chromosome  “inactive” gene: promoter is bound by nucleosomes and inaccessible o Activating transcription requires rearranging nucleosomes to expose promoter  Modification to histone proteins “tails” (extend from nucleosome) o Acetylation: acetyl groups (acid) partially neutralize histones (basic); promoter looser, open configuration o Histone acetyl transferases (HATs) (family of acetylating enzymes)  Open configuration spreads: HATs bind to acetylated histones and acetylate neighboring histones o Methylation: more complex. Effect on gene expression can be positive (activating) or negative (repressing)  Heterochromatin: dense, darkly-staining chromosome regions. Visible in mitotic chromosomes and sometimes interphase nuclei o Chromatin in heterochromatic regions is densely packed and not transcribed o Consequences of gene silencing in heterochromatin  Centromeres: no consequences. Don’t contain any genes  Mammalian X: equalizes gene dose  Euchromatin: the rest of the genome, i.e., not heterochromatin The types of cis-acting sequences  Cis-acting sequences: DNA sequences to which proteins bind to regulate transcription  Two classes: o Promoter (AKA basal promoter): near transcription start site  Major common feature: TATA box = sequence TATATAA. 25-30 base pairs upstream from transcription start site o Enhancers: variable in position and distance. Can be thousands of base pairs away from gene. Upstream or downstream, or in introns. A gene may have one or many. Function not dependent on distance or orientation from gene Types of transcription factors, their interactions with cis-sequences and their functions  Transcription factors: proteins that regulate transcription by binding to cis-sequences o Basal factors: bind to the promoter o Enhancer-binding transcription factors:  Activators (positive regulation)  Repressors (negative regulation)  Complex of basal factors binds to the promoter and recruits’ RNA polymerase o TATA-binding protein (TBP) o TATA-associated factors (TAFs) o Also mediator proteins  Basal factors are generic and ubiquitous. Not responsible for turning genes on/off How enhancers are identified.  Complexity of eukaryotic transcription regulation is largely due to activator & repressor binding to enhancers  Enhancers can be far away from gene regulated: DNA flexibility brings activator/repressor proteins in contact with basal factors  How do activators activate? o Recruit basal factors to the promoter. Stabilize binding of RNA polymerase o Remodel chromatin to expose the promoter  How do repressors repress? o Recruit corepressor proteins that interfere with the basal complex o Recruit corepressor proteins that close chromatin structure o Interfere with and “neutralize” activators  Compete for enhancer sites  Bind to and quench activator function  Bind to activator (sequester) to prevent DNA binding  Trans-acting factors o Transcription factors usually have three functional domains:  DNA binding domain: responsible for DNA sequence-specific binding  Dimerization domain: allows homo- or hetero-dimers. Most transcription factors act as dimers  Activation domain: interacts with basal transcription complex  How are enhancers identified? o Enhancers may be far away from regulated gene, at 3’ end of gene (or within introns) o Using recombinant DNA methods, the spacer DNA is cut into pieces, and each piece joined to a basal promoter and reporter gene  Reporter gene: a gene that makes a distinctive, easily identifiable product not normally present in the organism of interest  LacZ o -gal not present in most eukaryotic cells o Synthetic substrate X-Gal makes detection easy  Green fluorescent protein (GFP) o Can be detected in living cells o Insert each hybrid DNA construct into genome of the experimental organism o Result: some constructs show -gal expression while others do not o Conclusions: regions that do not contain enhancers show no expression of reporter genes. Regions that contain enhancers act in positive regulation and drives expression in the wing o Reporter gene expression driven by specific enhancers:  LacZ reporter gene expression in Drosophilia and mouse embryos Combinatorial control of gene expression.  Eukaryotic gene expression is combinatorial: depends on combinations of transcription factors  Genes are expressed differentially because different cell types have different combinations of transcription factors  Example: gene transcribed in skin and eye cells have enhancers for each tissue o Eye cells contain appropriate activators in S phase but no G1 o Skin cells contain appropriate activators o Liver cells don’t contain the correct combination and don’t express  Different cells have different combinations of transcription factors because… o Of earlier gene expression (transcription factors are proteins produced by earlier gene expression) o Protein modification in response to cellular conditions (phosphorylation due to a signal transduction pathway; steroid hormone binding) Post-transcriptional regulation  Splicing o Regulation by splicing:  Differential RNA splicing produces different mRNAs from the same primary transcript  Regulation of mRNA stability and translation o Small RNAs regulate mRNA stability and translation o 3 families of small RNAs, differing in deta
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