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Unit IX- Cell Cycle, DNA & Biotechnology Chapters 9 & 13 The Cell Cycle CH 9 Why make new cells at all?: Functions of Cell Division: Reproduction Functions of Cell Division: Growth & Development Function of Cell Division: Tissue Renewal Eukaryotic Chromosomes What is a “chromosome”? How related to a “gene”? How related to “DNA”? Cookbook analogy – go! Eukaryotic Chromosomes Chromosomes must duplicate so that each new cell has an exact copy of all of the code The two “copies” are called “sister chromatids” The Cell Cycle I-P-M-A-T-C I pee mainly after the commercials Interphase (90% of time) The DNA is in the THIN form called CHROMATIN during interphase •G1 – cell grows •S – sisters form; chromosomes copy •G2 – cell grows Mitosis (10% of time) • Pro – chromosomes form; nucleus goes away • Meta - middle • Ana – sisters apart • Telo – two nuclei form (on far sides) Cytokinesis – (NOT part of mitosis!) cytoplasm splits; cell membrane pinches in I-P-M-A-T-C I pee mainly after the commercials I-P-M-A-T-C I pee mainly after the commercials Which Phase? Which Phase? Hint: nuclear membrane dissipating Which Phase? Hint: cannot see the chromosomes Which Phase? Hint: “late ___” The Mitotic Spindle Kinetochore Attachment site between spindle fiber and sister chromatid The Mitotic Spindle Microtubules shorten from the kinetochore side Cleavage Furrow in Animal Cell Cell Plate in Plant Cells Which phases do you see? A D B E C Name the Labeled Structure: D A E B C F Name the Labeled Structure: microtubule sister chromatids metaphase plate kinetochore centrosome centriole Binary Fission (bacteria) Mitosis in eukaryotes may have evolved from binary fission in bacteria Evidence for Evolution of Mitosis Nucleus intact; spindle passes thru Nucleus intact; spindle within Spindle forms outside nucleus; nucleus breaks down The End – Part 1 Evidence for Chemical Signals to Control Cell Cycle 2 cells fuse…if one in M, other induced to start M Cell Cycle Control System Cell cycle proceeds step-wise But there are several regulation points (shown in red) Cell Cycle Control • Signals transmitted by “transduction pathways” (cell signaling!!!) • Animal cells tend to have built in stop signals …must wait for reports on cellular surveillance mechanisms • If do not pass G1…goes to G0 (most cells actually here) – nerve & muscle cells that generally don’t divide – some can be called back up for service 2 Molecules that Regulate Cell Cycle: • Protein kinases – Activate other proteins by phosphorylating them – Involved in both G1 & G2 checkpoints – Present at constant concentration in cells, but only activated when attached to a cyclin – Called “cyclin-dependent kinases” (Cdks) • Cyclins – Concentration fluctuates cyclically within cell – Concentration rises in interphase…drops in M Molecular Control at G2 Checkpoint MPF = “maturation-promoting factor” or “M-Phase promoting factor” Cyclins join up with Cdk’s; the MPF allows the start of mitosis Also causes break down of nuclear envelope (by phosphorylating its proteins) Later, MPF turns itself off by destroying the cyclins Cdk stays in cell inactive Internal Signals • Messages from Kinetochores – M checkpoint – Anaphase does not start until all chromosomes properly attached…why? – Signal from kinetochores delays anaphase – When all attached, “anaphase-promoting complex” (APC – a different one!) activates • Triggers breakdown of proteins holding sister chromatids together • Triggers breakdown of cyclin External Signals • Growth Factors – Proteins from certain cells that signal others to divide – Example: platelet-derived growth factor (PDGF) • Required to make fibroblasts (connective tissue) • At an injury, PDGF released…more tissue made – Probably specific GF’s for each cell type External Signals • Growth Factors – Density-dependent inhibition • Crowded cells stop dividing…(lack GF & nutrients) – Most animal cells exhibit anchorage dependence • Must be attached to lower layer to divide Effect of Growth Factor on Cell Division Describe how each regulates the cell cycle… • Cyclins & Cdks • Kinetochore Messages • Growth Factors Cancer Cells Normally, damaged cells undergo apoptosis… programmed cell death Cancer cells just stay damaged Cancer Cells Ignore Controls Cancer cells do not respond to normal control mechanisms (divide excessively) Perhaps have own GF or do not need GF or have short-circuit in pathway Cancer Cells • Cancer cells stop dividing at random stages in cycle • Can divide indefinitely if have nutrients • General process: – Transformation- change normal to cancer cell • Alteration of genes that control cell cycle – Tumor – if evades immune system; grows – Metastasis – malignant tumor; travels to other sites p53 (tumor suppressor gene) • Signals repair processes OR • Signals apoptosis Metastasis of a Malignant Tumor The End – Part 2 Lab - Mitosis Rate I. Purpose Gibberellic acid Phosphorus Willow extract Auxin Nitrate Aspirin – How does ____ affect the rate of mitosis in onion roots? II. Background – Discuss the chemical that your group will test. • You will likely need to look up info on your chemical. – Explain how root length is related to mitosis rate. Lab - Mitosis Rate Chemicals Tested – Gibberellic acid – Auxin – Phosphorus – Nitrate – Willow extract – Aspirin Lab - Mitosis Rate II. Background – Discuss the chemical that your group will test. • You will likely need to look up info on your chemical. – Explain how root length is related to mitosis rate. III. Hypotheses – State your null hypothesis. – Then, list each of the 6 chemicals being tested and rank from 1-6 in terms of which roots will have the most growth. (1 = most growth) Gibberellic acid Phosphorus Willow extract Auxin Nitrate Aspirin Lab - Mitosis Rate III. Hypotheses – State your null hypothesis. – Then, list each of the 6 chemicals being tested and rank from 1-6 in terms of which roots will have the most growth. (1 = most growth) IV. Procedure Gibberellic acid Phosphorus Willow extract – State your IV and DV. – Describe how you will obtain your data. – Sketch and label the set-up. • It should be clear enough that any random person could look at it and tell what you did. • NO step-by-step instructions!!! Auxin Nitrate Aspirin Lab - Mitosis Rate V. Data – Record root lengths (in mm) on data table – Show your calculations for both SD and SEM • no credit without work shown • Record all values to hundredths place • May skip “middle column” (x-x) – Create summary data table for your group data • Use proper IV/DV format Lab - Mitosis Rate V. Data Measure at least 8 roots, no more than 10 Be consistent with your cuts & measurements You may measure on the plant, then slice off root, or slice root first followed by measuring Record to nearest whole mm (no decimals) Place onions back in containers when finished (do not throw onions or growth medium out) Values for Roots in Water x 5 6 5 5 8 4 5 7 6 6 (x-mean)2 Mean = 5.7 .49 .09 .49 .49 5.29 2.89 .49 1.69 .09 .09 n = 10 Sum (x- )2 = 12.10 You calculate… SD and SEM Lab - Mitosis Rate V. Data – Create summary data table…use proper IV/DV format – Calculate SD for both. Show work. – Calculate SEM for both. Show work. – Graph mean growth for each medium. Include the error bars (2xSEM up & down) Lab - Mitosis Rate VI. Conclusion – Follow the “normal” format – 5 things! Write as many factual sentences as possible based on this information. • Start all sentences with “Cdk…” (use Cdk as the subject of your sentences) • Number each sentence. Warm-Up in your notes: 1. Draw and label a nucleotide. 2. Why is DNA a double helix? 3. What is the complementary DNA strand to: DNA: A T C C G T A T G A A C Warm-Up 1. What was the contribution made to science by these people: A.Hershey and Chase B.Franklin C.Watson and Crick 2. Chargaff’s Rules: If cytosine makes up 22% of the nucleotides, then adenine would make up ___ % ? 3. Explain the semiconservative model of DNA replication. Warm-Up 1. What is the function of the following: A. Helicase B. DNA Ligase C. DNA Polymerase (I and III) D. Primase E. Nuclease 2. How does DNA solve the problem of slow replication on the lagging strand? 3. Code the complementary DNA strand: 1. 3’ T A G C T A A G C T A C 5’ 4. What is the function of telomeres? THE MOLECULAR BASIS OF INHERITANCE Chapter 13 What you must know • The structure of DNA. • The major steps to replication. • The difference between replication, transcription, and translation. • The general differences between the bacterial chromosome and eukaryotic chromosomes. • How DNA is packaged into a chromosome. PROBLEM: Is the genetic material of organisms made of DNA or proteins? Frederick Griffith (1928) Frederick Griffith (1928) Conclusion: living R bacteria transformed into deadly S bacteria by unknown, heritable substance Oswald Avery, et al. (1944) – Discovered that the transforming agent was DNA Hershey and Chase (1952) • Bacteriophages: virus that infects bacteria; composed of DNA and protein Protein = radiolabel S DNA = radiolabel P Hershey and Chase (1952) Conclusion: DNA entered infected bacteria DNA must be the genetic material! Edwin Chargaff (1947) Chargaff’s Rules: • DNA composition varies between species • Ratios: – %A = %T and %G = %C Rosalind Franklin (1950’s) • Worked with Maurice Wilkins • X-ray crystallography = images of DNA • Provided measurements on chemistry of DNA James Watson & Francis Crick (1953) • Discovered the double helix by building models to conform to Franklin’s X-ray data and Chargaff’s Rules. Structure of DNA DNA = double helix – “Backbone” = sugar + phosphate – “Rungs” = nitrogenous bases Structure of DNA Nitrogenous Bases – – – – Adenine (A) Guanine (G) Thymine (T) Cytosine (C) purine pyrimidine • Pairing: – purine + pyrimidine – A=T – GΞC Structure of DNA Hydrogen bonds between base pairs of the two strands hold the molecule together like a zipper. Structure of DNA Antiparallel: one strand (5’ 3’), other strand runs in opposite, upside-down direction (3’ 5’) DNA Comparison Prokaryotic DNA Eukaryotic DNA • • • • • • • • • • • Double-stranded Circular One chromosome In cytoplasm No histones Supercoiled DNA Double-stranded Linear Usually 1+ chromosomes In nucleus DNA wrapped around histones (proteins) • Forms chromatin PROBLEM: How does DNA replicate? Replication: Making DNA from existing DNA 3 alternative models of DNA replication Meselson & Stahl Meselson & Stahl Replication is semiconservative Major Steps of Replication: 1. Helicase: unwinds DNA at origins of replication and creates replication forks 2. Initiation proteins separate 2 strands forms replication bubble 3. Primase: puts down RNA primer to start replication 4. DNA polymerase III: adds complimentary bases to leading strand (new DNA is made 5’ 3’) 5. Lagging strand grows in 3’5’ direction by the addition of Okazaki fragments 6. DNA polymerase I: replaces RNA primers with DNA 7. DNA ligase: seals fragments together 1. Helicase unwinds DNA at origins of replication and creates replication forks 3. Primase adds RNA primer 4. DNA polymerase III adds nucleotides in 5’3’ direction on leading strand Replication on leading strand Leading strand vs. Lagging strand Okazaki Fragments: Short segments of DNA that grow 5’3’ that are added onto the Lagging Strand DNA Ligase: seals together fragments Proofreading and Repair • DNA polymerases proofread as bases added • Mismatch repair: special enzymes fix incorrect pairings • Nucleotide excision repair: – Nucleases cut damaged DNA – DNA poly and ligase fill in gaps Nucleotide Excision Repair Errors: – Pairing errors: 1 in 100,000 nucleotides – Complete DNA: 1 in 10 billion nucleotides Problem at the 5’ End • DNA poly only adds nucleotides to 3’ end • No way to complete 5’ ends of daughter strands • Over many replications, DNA strands will grow shorter and shorter Telomeres: repeated units of short nucleotide sequences (TTAGGG) at ends of DNA • Telomeres “cap” ends of DNA to postpone erosion of genes at ends (TTAGGG) • Telomerase: enzyme that adds to telomeres – Eukaryotic germ cells, cancer cells Telomeres stained orange at the ends of mouse chromosomes Telomeres & Telomerase http://media.pearsoncmg.com/bc/bc_0media_bio/bioflix/bioflix.htm?8a pdnarep BIOFLIX: DNA REPLICATION Closing Questions 1. 2. 3. 4. What is recombinant DNA? What are plasmids? What are restriction enzymes (RE)? When DNA is cut using an RE, describe the ends of the DNA fragments. Closing Question A bacterial plasmid is 100 kb in length. The plasmid DNA was digested to completion with 2 restriction enzymes in 3 separate treatments: EcoRI, HaeIII, and EcoRI + HaeIII (double-digest). The fragments were separated by gel electrophoresis below. Draw a circle to represent the plasmid. On the circle, construct a labeled diagram of the restriction map of the plasmid. Closing Questions 1. Describe how a plasmid can be genetically modified to include a piece of foreign DNA that alters the phenotype of bacterial cells transformed with the modified plasmid. 2. How can a genetically modified organism provide a benefit for humans and at the same time pose a threat to a population or ecosystem? Biotechnology Terms you Must Know 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Genetic engineering Biotechnology Recombinant DNA Gene cloning Plasmid Restriction enzymes Sticky ends DNA ligase Cloning vector Nucleic acid hybridization PCR 12.Gel electrophoresis 13.RFLPs 14.Genomic library 15.cDNA library 16.DNA microarray assays 17.SNPs 18.Stem cells 19.Gene therapy 20.Transgenic animals 21.GMO (genetically modified organism) What You Must Know: • The terminology of biotechnology. • How plasmids are used in bacterial transformation to clone genes. • The key ideas that make PCR possible and applications of this technology. • How gel electrophoresis can be used to separate DNA fragments or protein molecules. • Information that can be determined from DNA gel results, such as fragment sizes and RFLP analysis. Terminology • Genetic Engineering: process of manipulating genes and genomes • Biotechnology: process of manipulating organisms or their components for the purpose of making useful products. • Recombinant DNA: DNA that has been artificially made, using DNA from different sources – eg. Human gene inserted into E.coli • Gene cloning: process by which scientists can product multiple copies of specific segments of DNA that they can then work with in the lab Tools of Genetic Engineering • Restriction enzymes (restriction endonucleases): used to cut strands of DNA at specific locations (restriction sites) • Restriction Fragments: have at least 1 sticky end (single-stranded end) • DNA ligase: joins DNA fragments • Cloning vector: carries the DNA sequence to be cloned (eg. bacterial plasmid) Using a restriction enzyme (RE) and DNA ligase to make recombinant DNA Gene Cloning Applications of Gene Cloning Techniques of Genetic Engineering Techniques of Genetic Engineering Transformation: bacteria takes up plasmid (w/gene of interest) PCR (Polymerase Chain Reaction): amplify (copy) piece of DNA without use of cells Gel electrophoresis: used to separate DNA molecules on basis of size and charge using an electrical current (DNA + pole) DNA microarray assays: study many genes at same time PCR (Polymerase Chain Reaction): amplify (copy) piece of DNA without use of cells Gel Electrophoresis: used to separate DNA molecules on basis of size and charge using an electrical current (DNA (+) pole) Gel Electrophoresis Microarray Assay: used to study gene expression of many different genes DNA microarray that reveals expression levels of 2,400 human genes Cloning Organisms • Nuclear transplantation: nucleus of egg is removed and replaced with nucleus of body cell Nuclear Transplantation Problems with Reproductive Cloning • Cloned embryos exhibited various defects • DNA of fully differentiated cell have epigenetic changes Stem Cells • Stem cells: can reproduce itself indefinitely and produce other specialized cells – Zygote = totipotent (any type of cell) – Embryonic stem cells = pluripotent (many cell types) – Adult stem cells = multipotent (a few cell types) or induced pluripotent, iPS (forced to be pluripotent) Embryonic vs. Adult stem cells Using stem cells for disease treatment http://learn.genetics.utah.edu/content/stemcells/sctypes/ Interactive: Go Go Stem Cells Applications of DNA Technology 1. 2. 3. 4. 5. 6. Diagnosis of disease – identify alleles, viral DNA Gene therapy – alter afflicted genes Production of pharmaceuticals Forensic applications – DNA profiling Environmental cleanup – use microorganisms Agricultural applications - GMOs Gene therapy using a retroviral vector “Pharm” animal: produce human protein secreted in milk for medical use DNA Fingerprinting RFLPs (“rif-lips”) • Restriction Fragment Length Polymorphism • Cut DNA with different restriction enzymes • Each person has different #s of DNA fragments created • Analyze DNA samples on a gel for disease diagnosis • Outdated method of DNA profiling (required a quarter-sized sample of blood) RFLPs – Disease Diagnosis VIDEO: INTRODUCTION TO DNA FINGERPRINTING Naked Science Scrapbook STR Analysis • STR = Short Tandem Repeats • Non-coding DNA has regions with sequences (2-5 base length) that are repeated • Each person has different # of repeats at different locations (loci) • Current method of DNA fingerprinting used – only need 20 cells for analysis STR Analysis STR Analysis Genetically Modified (GM) organisms • Organisms altered through recombinant DNA technology • Insert foreign DNA into genome or combine DNA from different genomes Biotechnology Techniques How to make Recombinant DNA Summarize: What is this technique? Draw and label a diagram to show this technique What are the main tools or materials involved? Applications: What is this being used for? Gel Electrophoresis PCR