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Genome Control Bacterial chromosome • • • • • • 1 circular chromosome Virus < bacteria < eukaryotes (# of bases) Nucleiod region Replication proceeds in both directions Rapid reproduction Single cell to colony (10^7 to 10^8 bacteria) within 12 hours • Plasmids • Variation in bacteria? Transduction: Phage carries genes from one bacterium to another Defective phage Conjugation: And you thought bacteria couldn’t have sex? F factor Exist as part of chromosome (episome) or as a plasmid (contains mostly genes for making sex pili F+ F+ and F+ F- F- and FDuring conjugation F+ F+ F- F+ Hfr – F factor on chromosome Genes transferred depends on initiation and termination R plasmids • Function of natural antibiotics? • Advantage of antibiotic resistant genes • Consequence of prescribing antibiotics? Transposable elements Recombination within a genome (between chromosome and plasmid or between plasmids • Insertion sequences – Only in bacteria – Transposase recognizes the inverted repeats – May disrupt coding sequence or regulatory genes • Transposons – More complex (extra genes) – Can add antibiotic resistance genes to plasmi already carrying genes. How would this spread? – Found in eukaryotic genomes as well Transformation • Uptake of exogenous DNA, resulting in newly acquired traits • Must be in competency (state able to take in DNA facilitated by membrane bound proteins • Competency is induced in E. Coli with CaCl2, MgCl2, or RbCl, and sudden changes of heat and cold (makes cell membrane permeable) • How do the Griffith and Avery experiments relate to transformation? Transformation efficiency • Amount of cells transformed/μg DNA • Through selection, colonies are counted • Each colony grew from a transformed cell pUC8 Plasmid • Present in E. Coli • Replicates independent of bacterial chromosome • Genetically engineered – Lac Z gene (β galactosidase) – MCR (can facilitate insertion of DNA (not required for replication) pUC8 plasmid • With Inducers (IPTG and X-Gal) colonies of bacteria appear blue – Lac Z β galactosidase X-Gal cleaved blue product • With inserted gene, lac Z is interrupted and colonies appear white – No Lac Z No β galactosidase X-Gal not cleaved white product pUC8 plasmid • Engineered E. Coli cells only synthesizes carboyxl terminal of β galactosidase protein • pUC8 plasmid contains gene for amino terminal • If pUC8 transforms cells, gene is fully functional Further selection • E. Coli NOT resistant to antibiotic ampicillin • pUC8 contains ampicillin resistant gene • The enzyme B-lactamase exits cell and inactivates ampicillin • Satellite colonies can appear around blue colonies (which color would they be , why?) Overview of Genome Control • Gene expression must respond to external cues • Differentiation allows cells to become specialized • Expression usually regulated at transcription level by DNA-binding proteins Genome packing (1st level) • Prokaryotic – Coiled and looped with protein (chromatin) • Eukaryotic – More complex – Highly condensed and coiled during metaphase around histones (4 + H1) – Histones leave only during DNA replication (what about transcription?) – Heterochromatin vs. euchromatin - Charged phosphates + charged a.a. Non histones Chromatin modification • Chromatin function – Compact DNA – Control transcription • Euchromatin vs. heterochromatin • Gene’s location relative to nucleosome and scaffold • DNA methylation – inactivates genes • Histone acetylation – increase gene expression by changing conformation of histones Distal Control elements (noncoding DNA) Proximal Recognizes TATA box -Transcription will occur, but inefficiently without activators -Repressors can bind to silencers to repress transcription (methylation) Recognizes proteins, including RNA Polymerase Transcription factors • DNA-binding domain (helix-turnhelix, zinc finger, leucine zipper) • Protein binding domain Coordinately controlled genes • Similar control elements before each gene allows simultaneous gene expression (analogous to operons) • steroid hormones and growth factors can act as signals to control expression Posttranscriptional control • Alternative splicing • mRNA degradation (hemoglobin example) – Poly(A) tail shortened 5’ cap removed mRNA degraded (enzymatic action) – mRNA stability controlled by part of sequence near 3’ end • Translational control - Preventing mRNA attachment to ribosome (could help store mRNA for later use) Post translational control • Protein modification – Cleavage – Chemical modification – Transportation • Posttranslational degradation Relevant tutorials • Campbell site – 19D, 19E, 19F • http://highered.mcgrawhill.com/sites/0072437316/student_view0/chapt er18/animations.html# – Transcription Complex and Enhancers (586.0K) – Control of Gene Expression in Eukaryotes (959.0K) Cancer caused by genetic changes All result in oncogenes “cancer genes” Agents of cancer – Spontaneous mutation, chemical carcinogens, radiation, viruses Signaling pathway that regulates cell growth oncogene Mutated tumor suppressor Multiple mutations leading to cancer Monday – drip Oxaliplatin Tuesday - 1/2 hour drip 5-fluorouracil Take home and use leucovorin for 22 hours Organization at the DNA level • Prokaryotic – DNA codes for protein, tRNA, or rRNA – Noncoding regions: Regulatory sequences – Coding sequence along a gene is continuous • Eukaryotic – Only 3% coding region – Noncoding regions: Regulatory regions, repeated sequences, introns – Multigene families Repetitive DNA - Different nucleotide composition, making the density distinct (appears as a satellite band) - Fragile X, Huntington’s - Near centromeres, telomeres Alu elements -5% of genome -300 pairs long -Transcribed into RNA - Unknown function Gene Families -Almost like extensive repetitive DNA -Some consist of identical genes, tandemly clustered -Most code for RNA -lack regulatory sequences -Higher affinity for oxygen -Transcription separated temporally Gene amplification • Higher rate of transcription during early development Genome rearrangement • • • • • Transposons - Stretches of DNA that can move within a genome Can interrupt gene expression Can carry a gene that becomes expressed when inserted next to a promoter Retrotransposon – needs RNA intermediate (Alu elements) 50% of maize, 10% of humans Relevant tutorials • Campbell site – 19A, 19B Problems cloning DNA • Getting cloned eukaryotic gene into a prokaryotic setting • Introns • Eukaryotic versus prokaryotic genome control • Engineer cloning vector to include prokaryotic promoters • Extract mature mRNA and make cDNA • Use yeast cells, artificial chromosomes VNTRs (type of DNA polymorphism) http://www.lsic.ucla.edu/ls3/tutorials/gene_cloning.html PCR of VNTRs • Extract DNA from sample • Lyse cell membranes to release DNA • Add nucleotides, primers, Taq polymerase to DNA sample • Thermocycler amplifies DNA • Gel electrophoresis of amplified DNA Sample results ladder More repeats homozygous heterozygous • Will this be enough evidence to convict someone matching the DNA sample? Mapping a genome • Linkage mapping – Based on recombinant frequencies • Physical mapping (chromosome walking) • DNA sequencing Comparison of methods Relevant tutorials • Campbell site – 19G, 20F • http://highered.mcgrawhill.com/sites/0072437316/student_view0 /chapter20/animations.html – How Tumor Suppressor Genes Block Cell Division (694.0K) Group presentations • 1 – Genomic and cDNA libraries (367368) • 2 – Gel Electrophoresis and RFLPs (372-374) • 3 – Southern Blotting (372, 373, 375) • 4 – Sanger Method (378) • 5 – DNA microarray assays and in vitro mutagenesis (379) Relevant tutorials • Campbell site: 20B, 20D, 20F • http://highered.mcgrawhill.com/sites/0072437316/student_ view0/chapter16/animations.html Genomic and cDNA libraries Gel Electrophoresis and RFLPs Southern Blottting Sanger Method DNA microarray assays and in vitro mutagenises