Study Guide - Chapter 18-25 PDF

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Study Guide - Chapter 18-25...

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FINAL EXAM MATERIAL Chapter 18 Regulation of Gene Expression a. Mechanisms of regulation Major Topics  Regulation of prokaryotic transcription  Regulation of eukaryotic transcription  Post-transcriptional eukaryotic regulation Gene Regulation Overview  Gene expression = gene to final product, all steps o Gene ฀ mRNA ฀ protein  Differential gene expression: each cell expresses only a subset of its genes. Typical human cells expresses ~20% of its genes o Cell types are different because they express different subsets of their genes o Some genes are expressed by many/ all cell types (eg., ATP synthase) o Some genes are expressed by a single cell type (eg., globin, insulin)  Gene regulation: mechanism of differential gene expression  Steps at which gene expression is regulated (in both proks and euks) o *Transcription o Splicing (in euks only) o Translation o mRNA stability o Protein stability *Most important mechanism Regulation of Bacterial Transcription TRP Operon  All living organisms require the amino acid tryptophan  The bacterium E. coli can make tryptophan from a precursor molecule in 3 steps  However if tryptophan is available in the environment the enzymes are not expressed  The three trp enzymes are the products of 5 genes (two enzymes are dimer proteins)  The 5 genes are adjacent and transcribed as a single long mRNA  Operon: cluster of bacterial genes with similar functions. Operon is transcribed as one long mRNA, so all genes turn on/off at the same time and equal amounts of protein are made  Trp operon: cluster of 5 genes for tryptophan synthesis  Trp operon is transcribed when tryptophan is absent, but not when tryptophan is present  When tryptophan is available the trp operon is repressed (turned off; no need to make tryptophan), but turned on when tryptophan is absent  Mechanism of repression: a protein repressor produced by the trpR gene binds to the operator, a DNA sequence at the beginning of the operon, blocking transcription  Tryptophan is a co-repressor: binds to the protein repressor

o Tryptophan bound to repressor ฀ repressor is active ฀ repressor binds to operator ฀ no transcription o No tryptophan ฀ repressor is inactive ฀ operon is transcribed Regulatory Proteins  General mechanism of transcriptional regulation: A regulatory protein(s) binds to a sequence in the DNA molecule near the promoter. Two types: o Negative regulation: binding of the regulatory protein prevents transcription  Example: trp repressor regulation of trp operon o Positive regulation: binding of the regulatory protein(s) is necessary for transcription Lac Operon  Similar system: lac operon  Lac operon encodes enzymes that break down lactose (milk sugar), a secondary energy source o “Preferred” energy source is glucose  Lac operon is active when: o Glucose is absent o Lactose is present  When glucose is absent, lactose turns on expression of lac operon Regulation of Eukaryotic Transcription  Eukaryotic genes are more complex and gene regulation involves occurs at multiple levels Transcription Factors and Control Elements  Transcriptional regulation: o DNA-binding proteins (transcription factors) bind to control elements (DNA sequences) and activate transcription (positive) or repress (negative)  Control elements may be close to or far away from the gene being regulated o Close: proximal control elements and promoter o Distant: distal control elements = enhancers. May be 1000s of base pairs from the gene  Transcription factor proteins bind to control elements to promote transcription: o General transcription factors and mediators bind to proximal control elements and the promoter o Activators bind to enhancers  Proteins bound to enhancers make contact with and stimulate RNA polymerase via looping of the intervening DNA  Differential gene expression is due to specific combinations of activator proteins Combinatorial Control  Cell-specific transcription results from different combinations of transcription factors. Examples: o Liver cell contains red, yellow and grey trxn factors o Lens cell contains orange and gray trxn factors o Expression of albumin vs. crystalline gene is due to different combinations of trxn factors

Why do liver cells contain red and yellow but not orange trxn factors? Trnx factors are proteins, and thus produced by transcription & translation Differential presence of trxn factors is due to earlier differential gene expression Different cell types come through a sequence of differential expression of transcription factors  Last “generation” of trxn factor turns on genes that encode specialized proteins Chromatin Structure  Chromatin: combination of DNA + proteins o Histones: small basic proteins o DNA wraps around histone complexes, coils up into compact structures (much shorter) o Does chromatin regulate transcription?  Chromatin structure is regulated by chemically modifying histones and DNA o Histone acetylation. Addition of acetyl groups (acid) promotes a “looser” chromatin structure & promotes gene expression o DNA methylation. Methyl groups added to base C. Represses transcription  Modifications and patterns of gene expression are passed down at cell division to descendent cells = epigenetic inheritance Post-transcriptional Eukaryotic Regulation Alternative Splicing  Alternative splicing: primary transcript may be spliced in different ways o Example: two alternative troponin mRNAs with different exons. Different proteins will be translated from these mRNAs miRNAs and siRNAs  Small RNAs may regulate mRNA stability and translation  Non-coding RNAs: do not code for protein sequences. o miRNAs (micro RNAs) and siRNAs (short interfering RNAs). Both are 20-23 nt RNA molecules o Complementary to some mRNAs. Binding of miRNA to target mRNA induces mRNA degradation or translation inhibition    

b. Development and Cancer Major Topics  Development  Cancer Development  The development of a multicellular organism with hundreds / thousands of cell types requires regulation of gene expression… o At the correct time  Eg., the crystalline gene is expressed only when eyes are developing o In the correct location  Eg., only the developing lens expresses crystalline  How do the simple instructions in DNA carry out this complex process? Determination and Differentiation

Cells have specialized functions because they express unique genes o Examples: liver cells – albumin; lens cells – crystalline; some pancreatic cells – insulin; red blood cells – globins  Differentiation: cellular specialization. Cell acquires its final specialized form  How does a cell in the eye “know” that that’s what it is, and that it should express the crystalline gene?  Determination: cells acquire information and become committed to a particular fate in the organism  Determination precedes differentiation o A cell commits to a particular fate (determination) and then acts on it (differentiation)  Determination is under genetic control  Example: determination of muscle cell identify reqs the expr of MyoD, a master regulatory gene o MyoD encodes a transcription factor protein that promotes the expression of other muscle-specific genes Pattern Formation  Since all cells have the same genes, how do cells in different parts of the body become different? o Spatial control of gene expression, determination and differentiation = pattern formation  Different patters arise because regulatory molecules are distributed asymmetrically  Asymmetric regulatory molecules: o Unequal cytokinesis and different cytoplasmic contents (molecules = cytoplasmic determinants). o Different extracellular signals (process = induction) Bicoid  Example of a cytoplasmic determinant: Drosophila bicoid mRNA and protein. mRNA localized at the anterior tip of the egg during oogenesis. Translated after fertilization  Bicoid protein = transcription factor. Early gene expression in the embryo results from concentrations of bicoid protein  Subsequent differential gene expression leads to the larval body pattern Homeotic Genes  Homeotic genes: “master genes” that control cellular identity o Homeotic proteins are transcription factors  Mutations in homeotic genes in Drosophila cause mistakes in segment identity (antenna ฀ leg)  Genetic hierarchies in early fruit fly development result in the proper expression of homeotic genes  Homeotic genes regulate the anterior-posterior development of all bilateral animals  Animals have a common evolutionary origin and genetic blueprint Cancer  Cancer: disease of unregulated cell proliferation  Mutations cause changes in cell behavior 

o Loss of inhibition of proliferation o Loss of cell adhesion – ability to leave tumor and move elsewhere o Ability to stimulate vascularization Genetic Control  Cell proliferation is genetically controlled through signaling processes… o Neighboring cells may stimulate or inhibit cell division  …and regulation of transcription o Entry into mitosis involves adoption of an internal genetic program  Cancer results when cells accumulate mutations that inactivate or override normal proliferative controls  Analogy o One controls a car by stepping on the accelerator pedal to go, and the brake to stop o Unfortunate things will occur if…  …the accelerator pedal is stuck, or  …the brakes fail  Two types of genes regulate normal cell division: o Proto-oncogenes encode proteins that promote cell division o Tumor-suppressor genes encode proteins that repress cell division  Both sets of regulatory controls are subject to mutation: o Oncogenes (mutant version of proto-oncogenes): Mutant proteins promote cell division, but lack the ability to be regulated o Mutant tumor suppressor genes encode defective proteins that do not repress cell division  Oncogene analogy: a “stuck accelerator pedal”  Tumor suppressor mutation analogy: “defective brakes”  Mutations in two types of genes result in cancer: o 1. Positive regulations of proliferation. Mutations can cause protein to get stuck in the “on” position. Result: inappropriate cell proliferation o Oncogene: a mutant allele of a normal cellular gene. One mutant allele is sufficient, ie., promotes cancer in a dominant manner o Proto-oncogene: normal allele of an oncogene o 2. Negative regulators of proliferation. Mutation alleles produce a non-functional protein that fails to act o tumor-suppressor genes: recessive; both genes must be mutant for cancer progression  Proto-oncogenes become oncogenes by: o Overexpression due to gene rearrangement, or gene amplification (too many copies) o Point mutations resulting in overexpression o Point mutations that cause too much activity or inactivate regulatory sites  Growth factor signaling pathway stimulates cell division  Step 3: G-protein Ras turns on and off in response to ligand binding to receptor

Mutant Ras is always active, even in the absence of the signal ฀ unregulated cell division ฀ cancer Learning Goals  What is gene regulation and why is it necessary?  Physiological regulation in bacteria o Concepts specific to bacterial gene regulation: operon, inducible, repressible o Logic of repressible trp operon regulation o Positive and negative regulation. Mechanisms of negative regulation of trp operon  Eukaryotic gene regulation o Levels are which regulation can occur o Mechanisms of transcriptional regulation: chromatin structure, transcription factors, promoters, enhancers, activators. Combinatorial control of gene expression o Post-transcriptional: regulation of splicing, mRNA and protein lifetime. Regulation my miRNAs and siRNAs  Mechanisms of cellular fate assignment – cytoplasmic determinants and extracellular signals  Bicoid as a determinant of embryonic asymmetry  Homeotic genes and cellular identity  Cancer – progression of cellular changes leading to tumor and metastasis o Oncogenes and proto-oncogenes and mode of action  Example of an oncogene o Tumor-suppressor genes and mode of action Chapter 19 Viruses 

Major Topics  Viral structure  Bacteriophages  Animal viruses  Odds and ends: treating viral diseases, virus-like particles, viral origins Viruses General  Virus: non-living obligate intracellular parasites o Only reproduce within living cells  All living organisms have viruses: prokaryotes and all varieties of eukaryotes  Viruses may have restricted host ranger or infect many related organisms Viral Structure  Capsid: protein shell that surrounds the genome o Many copies of one or a few different types of proteins o Many configurations & shapes o Some also have a membrane that surrounds the capsid, the viral envelope  Viral genetic material is diverse: double stranded DNA, ssDNA, dsRNA, ssRNA

Bacteriophages Lytic Cycle  Bacteriophage: viruses that infect bacteria  Lytic cycle (kills the host cell): o 1. Virus attaches to outside of host cell o 2. Genetic material is injected into cell o 3,4. Virus hijacks the bacterium’s transcription / translation machinery to make its proteins and replicate its genome o 5/ Bacterium is lysed and newly made phage particles are released to infect new cells Lysogenic Cycle  Lysogenic infection: phage exist within the host cell without reproduction or killing the host  Viral DNA is integrated within the bacterial chromosome and passed to progeny  Lysogenic phages can become lytic Animal Viruses  Diverse genome types and modes of infection  Many (but not all, such as Ebola) animal viruses infect only certain cell types. Examples: o Influenza virus: cells of the respiratory tract o Papovavirus: skin cells (warts) o HIV: particular types of T lymphocytes (white blood cells)  Animal viruses use a wide variety of DNA/ RNA molecules as genetic material Reproductive Cycle  Example: enveloped RNA virus (example: Ebola) o Envelope fuses with host plasma membrane, releasing genetic material into cell o ssRNA genome is used as template to make mRNA (by an RNA-dependent RNA polymerase) o mRNA is template for new RNA genomes o mRNAs are translated into capsid and envelope proteins, into rER/ Golgi system, ending up at cell surface o Capsid assembles around new RNA o Assembled viral particles are budded off with host plasma membrane surrounding them Ebola  Genome is a ssRNA molecules, 19 kbases  Contains 7 genes: o 1 gene for RNA-dependent RNA polymerase o 5 genes for capsid proteins o 1 gene for a regulatory protein  Natural hosts: bats, chimpanzees. Probably not seriously pathogenic in natural hosts o Probable spread to humans via chimp meat or eating fruit that was partially eaten by bats

o Infects many cells of the immune system, leading to immune system collapse. In later stages infects endothelial cells (line gut and blood vessels) leading to diarrhea and internal bleeding o Human-to-human transmission via body fluids: blood, feces, vomit. No known aerosol transmission HIV 

HIV, a retrovirus: ssRNA genome is copied into dsDNA by enzyme reverse transcriptase. DNA acts as template for synthesis of mRNA/ genome

Odds and Ends Why Are Viral Diseases Hard to Treat?  Antibiotics are effective against bacterial infection. Why aren’t they effective against viruses?  Antibiotics are selective poisons that inhibit bacterial metabolism. Do not affect eukaryotic metabolism o Examples: penicillin inhibits bacterial cell wall synthesis; tetracycline inhibits bacterial protein synthesis  Because viruses lack their own metabolism and have few of their own enzymes: they “borrow” host enzymes for replication, transcription, etc. Result: much smaller number of viral-specific functions to poison o HIV: reverse transcriptase and envelope protein processing o Herpesvirus: DNA replication o Ebola: no targets yet identified o Vaccine: a weakened/ inactivated virus is injected and body makes antibodies (provides immunity to future infection) Virus-like Particles  Viroids: circular RNA molecules (no capsid) that replicate in plants. Disrupt cell regulation (how?)  Prions: misfolded proteins that can induce the misfolding of other copies of the same protein o The causative agent of mad cow, and related diseases Viral Origins  Viruses are related in structure and sequence to other intracellular genetic “parasites”: o Transposable elements (euks), transposons (bacteria): segments of DNA that excise themselves from the chromosome and reinsert at another location o Plasmids: DNA circles that replicate independently of the host genome (bacteria and fungi)  Are viruses descendants of these, but have gained the ability to move from cell to cell? Or vice-versa? Learning Goals  General aspects of virus structure  The phage lytic and lysogenic cycles  Animal viruses and their reproductive cycles  Why viral diseases are hard to treat

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How viroids and prions differ and are similar to viruses Where viruses come from

Chapter 20 Biotechnology Major Topics  Recombinant DNA (methods)  Polymerase chain reaction  Applications of DNA biotechnology  Cloning organisms  Stem cells What is “Biotechnology”?  Biotechnology: methods used in medicine, pharmaceutical industry, forensics, etc.  Two (largely unrelated) methods: o DNA manipulation and gene analysis. Recombinant DNA, PCR, DNA sequencing, DNA hybridization o Stem cell and organismal cloning methods based on nuclear transplantation & cell manipulation Recombinant DNA  Recombinant DNA: replication of foreign DNA in a host organism. AKA DNA cloning o Host: usually a bacterium  Foreign DNA will be replicated if joined to bacterial DNA that o Has its own origin of replication and o Is beneficial to host (eg., antibiotic resistance)  Vector: bacterial DNA that is fused to foreign DNA molecule and causes it to be replicated o Most vectors are modified plasmids or bacteriophage Overview  Many bacterial species contain small circular accessory chromosomes, plasmids  In recombinant DNA expts, DNA from a foreign source is joined to a plasmid; foreign DNA replicated as cells divide, making many copies from 1 starting molecule o AKA “DNA cloning” or gene cloning  Plasmid here is a cloning vector Cutting and Joining DNA Molecules  Method of joining foreign and vector DNA: o Cut with restriction enzyme (cuts DNA at a specific sequence) o Mix molecules, which transiently join via sticky ends (complementary single stranded DNA) o Make new covalent bonds with DNA ligase Applications of DNA Biotechnology Expressing Eukaryotic Genes in Bacteria  Cloned euk genes may be expressed in bacteria. Problems:

o Bacterial and eukaryotic transcription and translation regulatory signals are different  Solution: use only the euk protein-coding region. Use bacterial regulatory sequences o Euk genes have introns; bacteria lack the ability to splice  Solution: clone cDNAs (copies of mRNAs), which lack introns  cDNA = complementary DNA. A DNA copy of mRNA (no introns in mRNA)  Synthesis: start with a population of mRNA  RNA is copied to DNA by enzyme reverse transcriptase o Reverse transcriptase makes DNA copies of ssRNA templates (purified from retroviruses)  cDNA is cloned like other DNA  Structure of a DNA clone that expresses euk protein in bacterial cell o Cloning vector o Bacterial transcription and translation signals o Eukaryotic cDNA Pharmaceuticals  Human protein produced in bacteria for medical uses: o Insulin o Human growth hormone o “Clot buster” drugs used to treat heart attack and stroke (TPA, streptokinase)  Other organisms can also be used as hosts for expressing pharmaceuticals o Goats and sheep (drug is in the milk) o Plants (large quantities are cheap to produce) Agriculture and Other Uses  Transgenic organism: an individual that contains a foreign gene or a gene altered in the lab o Soybeans & corn resistant to herbicides o Soybeans & corn resistant to insects (poisonous protein native to bacteria) o Rice that makes its own vitamin A  Also bacterium that digests waster Gene Therapy  Gene therapy: insert normal gene into cells to replace a mutant gene o Ie., patient is a “transgenic organism”  Problems: how to get genes inside cells? o Modified viruses o How to get genes to the correct cells?  Partial success: treatment of Severe Combined Immunodeficiency Syndrome (SCID) o Unexpected problem: high incidence of leukemia Forensics  DNA...