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Notes from lectures and readings for Exam 3. Arranged by lecture, concepts are color-coded and images are included to better illustrate certain concepts.
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Gene expression can be regulated at many steps, but in prokaryotes mostly at transcription. Constitutive genes: essential to normal, growing cell - always active/transcribed - “housekeeping” Regulated genes: products needed at specific times, activity controlled in response to the needs of the cell/organism - expressed in specific environmental/developmental context Mechanisms: repressor/activator proteins, attenuation, antisense RNA, riboswitch LAC OPERON - a cluster of genes coordinately regulated by one promoter Lactose induces expression of 3 genes. Beta-gal - lactose metabolism. Permease - brings lactose into the cell. Transacetylase - we don’t know. Polycistronic mRNA - proteins normally not always expressed, only needed when lactose is present - studied in mutant strains. Constitutive mutant e-coli strain - proteins produced all the time. Mutation site mapped to novel gene - controller of Inducibility - lac I. E - Coli merozygotes created to study the effects How do mutations in the I-gene affect b-gal production? - does it produce a trans-acting factor? I-gene encodes a protein that negatively controls b-gal gene - repressor normally blocks transcription, presence of inducer overcomes block. LAC OPERON FLOW CHART If P- -> no binding of Rna Pol 3 to DNA ---> If Z- -> no b-gal( Z encodes for b-gal). If Y- -> no b-gal (Permease allows lactose into the cell) Next…. If Oc - b-gal is always produced (constitutive operator, transcription is always on). Next…. If Is - b gal is not produced - the inducer doesn’t cause the repressor to unbind. Next... If I- , b gal is always produced, repressor can’t bind. Next... If I+ - everything is normal and b-gal is regulated. Nonsense mutations - sense codon is changed to nonsense codon - premature transcription termination, downstream products are not made. Missense mutation - one sense codon is swapped out for another - will not affect downstream products
Mutation - change from “wild type”. may cause phenotypic variation leading to disease or evolution Large scale (chromosomal) - change in structure or number - can be viewed under light microscope - deletion, inversion, duplication, translocation, entire chromosomes added or deleted or small scale (point) - 1 base pair replaced, added or deleted, missense, nonsense and frameshift - may be silent Transition - purine to purine (A->G, G->A) or pyrmidine to pyrimidine (C->T, T->C) Transversion - purine to pyrmidine, pyrimidine to purine (A->T, C->G, G->C, T->A) Missense mutation - replacement of one sense codon with another sense codon - neutral if does not affect function Nonsense - sense codon to nonsense codon - truncated, nonfunctional protein Silent - point mutation where the amino acid is the same due to degeneracy Frameshift - addition or deletion of multiples of 1 base pair - shifts frame so that sequence downstream is incorrect. Puts nonsense codon in frame so protein is shorter than normal. Somatic - in body cells, not heritable, can lead to cancer, not all cells have mutation. Germline - gametes, heritable, all cells in affected individual have mutation Spontaneous - occur during the lifetime of the cell - errors in replication, free radicals. DNA repair machinery can make mistakes - undetected incorrect insertions due to wobble pairing established by subsequent replication Frameshift mutations due to strain slippage - common at repetitive sequences Normal biochemistry leads to.. depurination(loss of a purine, common problem that is typically corrected - if not, replaced with a random base - typically a) Deamination (loss of amino group from cytosine(change to uracil, G/C -> A/T) and adenine (A/T -> G/C) - 2 rounds of replication Induced - exposure to external agents - base analogs, base modifiers, intercalating agents, UV light Ames Test - plate his- bacteria on medium lacking histidine w chemical, or chemical mixed with liver enzymes. If growth occurs, then it is a mutagen - more growth, stronger mutagen. Several different types of mutagens (chemicals that induce mutation) Base Analogues - chemicals with structures similar to normal bases, can be incorporated into DNA in s-phase - ionization changes basepair properties in subsequent rounds of replication. Base Modifiers - add or remove chemical groups from a base so that it will not pair properly with template Intercalating Agents - UV Light - causes adjacent pyrimidines to bind together so they will not basepair with complementary DNA strand during replication Intergenic Suppression nonsense mutation in gene for eye color, suppressed by gene encoding for tRNA Initial and suppressor mutation in protein subunits that break and then reestablish connections Intragenic Suppression - “2 wrongs make a right” - normal function stays intact. changing one base in a codon, then changing another to restore the original codon due to redundancy Changing one amino acid from negative to positive charge, then changing another from positive to negative - DNA Repair - require 2 strands, are redundant (more than one way to repair) Direct Repair - repairs thymine dimers - photolyase(light activated) + alkyl- and methyl-removing transferase enzymes Nucleotide Excision Repair - distortion recognized as an error - endonuclease activity breaks phosphodiester bonds on either side, gap filled by DNA Pol and sealed by DNA ligase Base Excision Repair - initial removal of base - DNA Glycosylase removes modified base, DNA endonuclease removes sugar, DNA Pol adds new nucleotide and DNA ligase seals the nick. Mismatch Repair - corrects mismatches during replication - new strand must be distingushable from old strand - differing methylation patterns - distortions cut out, DNA Pol resynthesizes, DNA ligase seals the nick.
Recombinant DNA technology - “genetic engineering” - region of DNA can be separated from genome for further study Gene of interest + promoter + regulatory sequence Restriction Enzymes - each enzyme recognizes a specific DNA sequence Restriction sites, 4-8 bp long, present randomly in genome, mostly palindromic. Make double-stranded cuts in DNA - a normal part of bacterial defence against infection T1 and T3 do not cut at restriction site - T2 do. Staggered cuts - generate cohesive “sticky ends” - complementary pieces can base-pair - DNA seals the deal Blunt cuts - leave no single-stranded overhangs for base-pairing Gel Electrophoresis - electricity in agarose gel causes DNA to migrate through it - smaller DNA moves further and faster How do we find a specific piece of DNA from the whole genome? Southern Blotting Probe (single-stranded piece of DNA complementary to the sequence of interest - labeled) The signal on paper corresponds to the size of fragments probe binding confirms the sequence of interest is present. Can be used to detect homologus genes in different species Fluorescence in situ Hybridization
Linkage Map - distance based on experimental data, subject to error - map units or centiMorgans Physical map - direct analysis of DNA - more precise than a genetic map - basepairs - kilobases and megabases The Sanger Method - DNA replication in vitro with ddNTPs (dideoxy nucleotides - no 3’ OH so prematurely terminate replication) Clone DNA to have enough material Synthesize a labeled primer (either fluorescent or radioactive) - if automated, use fluorescent - read automatically by laser and detector. denature DNA prepare ingredients for PCR + ddNTPs, Divide mix into four tubes - one type of ddNTP for each at a small concentration incubate at 37*c, Gel Electrophoresis Next-gen sequencing - millions of fragments at once, faster and cheaper, whole genomes of many individuals in days, several methods being worked on
A coordinated effort between 18 countries - how useful is it? VERY. Developing and improving the necessary tools Why was it so tough? Traditional methods allowed for sequencing a few thousand bp - chromosomes are tens of millions of base pairs. Chromosomes must be cut for cloning and sequencing - lots of repetitive sequences, so hard to determine the order. Public vs. Private Francis Collins and the NIH - DNA randomly selected from large collection - lots of labs using 600 sequencers. Craig Venter and Celera - mix of few individuals’ DNA - 1 large facility with 300 sequencers + a supercomputer Map-based approach - cut up chromosomes into pieces, create a genetic library, find the overlap (contig) Contig-map Making - a collection of clones with overlapping/contiguous pieces of DNA Chromosome-specific library, use genetic markers for organization Order of clones is determined based on the overlap of specific genetic markers -> DNA in each clone is cut into pieces, ligated into vectors and sequenced Logical, methodical, and proven - but SLOW, HARD, TIME-CONSUMING Shotgun Mapping - cut chromosomal DNA into small pieces - put pieces into vectors -> sequence all pieces use software to put pieces together in sequence Fast, easy, cheap - but INACCURATE sequence HGP gave a consensus sequence 5% of genetic material is coding 20K genes:500K proteins - doesn’t show differences Inspired the International HapMap project - charts where the genome differs (single-nucleotide polymorphisms) SNPs arise due to mutation, inherited like other alleles, do not often produce a phenotype Haplotype - set of SNPs on a single chromosome - can change by mutation or recombination GOAL - map disease alleles by comparing haplotypes of normal and infected humans Genome-wide scan - up to a million SNPs can be analyzed at once - using a DNA microarray looking for a probe - probe is immobilized and sample added - vs Southern blotting Genome-wide association studies - use SNPs throughout genome to find genes with interest Analyze and compare SNPs in those with and without the disease those more common in diseased invididuals are likely to be linked to a disease-causing mutation DNA fingerprinting - based on analyzing Short Tandem Repeats - STRs Also called microsatellites, STRs are short DNA sequences that repeat in tandem - many loci within the genome - number of tandem repeats varies among individuals compare lengths using PCR and electrophoresis - heterozygotes generate 2 different color bands FBI analyzes 20 STRs when identifying suspects - reduces the probability of misidentification
Regulation of gene expression is complicated in eukaryotes - DNA is organized into chromatin - nuclear envelope physically separates transcription and translation
Chromatin - diffuse during most of cell cycle - less compact than in metaphase Heterochromatin - tightly packed, inactive/few genes. Euchromatin - loosely packed, active/many genes. 1 - DNA packaging chromatin remodeling(repositioning histones, no chemical histone or chromosome modification), DNA methylation - CPG islands(CG repeats) common in promoters, correlates with decreased txn, correlates with deacetylation of histone proteins(which allows for tighter chromosome packaging) Histone modifications - amino acids in tail of protein - creates epigenetic mark, may increase or decrease txn
2. Transcription Initiation RNAP II transcribes pre-mRNA - promoters have several different cis-acting elements (TATA Box) Trans-acting factors bind to cis-acting elements. Activator proteins bind to enhancers, increase transcription. Repressor proteins bind to silencers, decrease transcription. Insulator-binders bind to insulators - establishes boundaries for enhancers. (insulators create loops in chromatin - enhancers effect genes in their loop) Transcription outline - transcription activators bind DNA and mediate transcription Several eukaryotic genes activate from the same stimulus, vs 1 stimulus to 1 gene - regulatory elements vary Example: regulated expression of metallothioneins