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Biology 102 Genetic Section Notes.docx

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BIOL 102

Biology 102 Study Notes Jennifer Williams Biology 102 - Genetics Section Table of Contents Chapter 10: Nucleic Acid Structure and DNA Replication Chapter 11: Gene Expression at the Molecular Level Chapter 12: Gene Regulation Chapter 13: Mutation, DNA Repair, the Cell Cycle, and Cancer Chapter 14: Eukaryotic Chromosomes, Mitosis, and Meiosis Chapter 15: Inheritance and Phenotype Determination Chapter 18: Genetic Technology Biology 102 Study Notes Jennifer Williams Chapter 10: Nucleic Acid Structure and DNA Replication What are Genes Made of?  DNA must be able to replicate itself  DNA must be able to control living processes **Original DNA was RNA, some scientists believe DNA: Early Evidence Scientist Experiment & Observations Friedeich Meischer Isolated a phosphate-containing acid from the nucleus: Nuclein from (1869) white blood cells (didn't know the structure) Griffith 1) Injected living type S (Smooth) bacteria into mouse - Mouse died 2) Injected living type R (Rough) bacteria into mouse - Mouse lived 3) Injected heat-killed S bacteria into mouse - Mouse lived 4) Injected living type R and heat-killed type S bacteria into mouse - Mouse died: Living type R cells have been transformed into virulent type S cells. Concluded that a transformation had occurred: the R bacteria took something up from the dead S: a "transforming principle" (molecule of some sort changed the R bacteria (into S)! 35 Hershey-Chase (1952) Found that the 32ended up in the supernatant (viral capsids) Found that the P ended up in the bacterial pellet Concluded that the transforming principle (genetic material) was DNA, and NOT a protein. **See Hershey and Chase Experiment on McGrawHill online: Evidence for DNA Structure Scientist Experiment & Observations Edwin Chargaff Molar quantity of Adenine=Thymine (1930/40s) Molar quantity of Cytosine=Guanine Each species has its own ATGC complement "Chargaff's Rules": always A=T; C=G Franklin & Wilkins X-Ray diffraction analysis of DNA showed: DNA was a linear (1940-1953) molecule; DNA was a helix because the diffraction pattern X. Biology 102 Study Notes Jennifer Williams James Watson and Discovered DNA - helped by Rosalind Franklin, who never knew that Francis Crick (1953) Watson and Crick has her X-ray photographs Franklin was not given credit, because she died before the Nobel Prize was given to Watson and Crick Key features of DNA:  Two strands of DNA form a double helix  The bases in opposite strange hydrogen bond according to the AT/GC rule  The 2 strands are anti-parallel (read 5' to 3') --> 5' end has a free carbon, 3' end has hydroxyl group  There are ~10 nucleotides in each strand per complete turn of the helix. DNA Structure  Bases are bound by hydrogen bonds (GC 3bonds, AT 2 bonds)  Phosphodiester bonds between deoxyribose sugar subunits  Phosphates are attached between the 5' and 3' carbons of the sugars  Length of DNA in one cell = 1m; Length of DNA in body = Earth to Pluto and back  Strands are of opposite polarity: antiparallel  Strands are hydrogen bonded via: A pairing with T; C pairing with G Purines = Double ring structure: Adenine and Guanine (Pure GAGA) Pyrimidines=Single ring structure: Thymine, Cytosine, Uracil DNA vs. RNA DNA (Deoxyribonucleic Acid) RNA (Ribonucleic Acid) Deoxyribose Sugar (1 less oxygen Ribose Sugar than ribose sugar) ATGC AUGC = Uracil instead of Thymine: Has 1 less CH than 3 Thymine Double stranded Single stranded Central Dogma of Molecular Biology DNA=>*transcription*=>RNA=>*translation (using messenger RNA)*=>Protein Biology 102 Study Notes Jennifer Williams **Note: In prokaryotes, transcription and translation occur in the cytoplasm; in eukaryotes, transcription occurs in the nucleus, which translation occurs in the cytoplasm DNA Replication Options 1) Semi-conservative mechanism: DNA replication produces DNA molecules with 1 parental strand and 1 newly made strand 2) Conservative mechanism: DNA replication produces 1 double helix with both parental strands, and the other with 2 new daughter strands. 3) Dispersive mechanism: DNA replication produces DNA strands in which segments of new DNA are interspersed with the parental DNA **DNA is replicated semi-conservatively (Option #1) through following experiment Meselsohn and Stahl  Bacteria take up any isotope of N to make DNA (in bases)  Used N (heavy N) - instead of N (normal N)- to make heavy DNA in Bacteria; several generations  Then provided with N-containing medium later generations of growth  DNA centrifuged with CsCl (dense element; easily soluble)  During centrifugation: CsCl makes a density gradient  The DNA moves to region of same density  Called isopycnic (same density) centrifugation  After 2 Rounds of replication, 1/2 light N, 1/2 heavy N was found. Only explained through semi- conservative mechanism, because conservative=always heavy bonds **See: McGrawHill video: DNA Replication - 3 Types: 1) Bidirectional replication in eukaryotes  DNA strands unwind at the origin of replication  DNA replication begins outward from the two replication forks  DNA replication continues in both directions 2) Single origin of replication in bacteria (prokaryotes)  DNA strands unwind, and DNA replication begins  DNA replication is completed 3) Multiple origins of replication in eukaryotes  DNA strands unwind, and DNA replication begins at multiple origins of replication  DNA replication is completed (Kinetochore proteins at the centromere) How DNA Replication Starts  Origin of replication - particular base sequence  Helicase unwinds DNA - travels along one DNA strand in the 5' to 3' direction and separates the DNA strands  Single strand binding proteins coat the DNA strands to prevent them from re-forming a double helix after Helicase has unwound the DNA  Replication starts in the replication fork  Excessive Coiling: opening double helix causes increased coiling of DNA further down stream Biology 102 Study Notes Jennifer Williams  Topoisomerases (enzymes) open DNA to allow uncoiling and then rejoin DNA. Travels slightly ahead of the replication fork and alleviates coiling caused by the action of Helicase  Now the DNA is ready for replication DNA Replication (And parts involved) - Also see diagram below for summary 1) RNA primer: is added first by the DNA primase. DNA primase makes RNA primers to begin the replication process 2) Subunits add at the 3' end: but new strands elongate in opposite directions 3) Leading strand: elongates into fork 4) Lagging strand: elongates away from fork 5) Elongation proceeds smoothly on leading strand 6) Addition to lagging strand is by 100-200 base Okazaki fragments 7) Lagging strand grows discontinuously because of the size of the Okazaki fragments (That's why it Lags!!) 8) DNA polymerase III makes DNA from the RNA primers. DNA primase hops back to the opening of the form and makes a second RNA primer for the lagging strand. **Movie: How Nucleotides are Added in DNA Replication: http://highered.mcgraw-  Two DNA Polymerases: Polymerase 1= removes RNA primer; Polymerase 3= binds and checks final DNA replication product What Happens at the Ends of Chromosomes: Telomeres  Specialized form of DNA replication only in eukaryotes in the telomeres  Telomeres are a series of repeat sequences within DNA and special proteins  Telomere at 3' does not have a complementary strand and is called a 3' overhang.  If Telomeres did not exist, each replication you would loose ends of chromosomes=loosing DNA! If this replication problem were not solved, linear chromosomes would become progressively shorter  DNA polymerase cannot copy the tip of the DNA strand with a 3' end: no place for upstream primer to be made Biology 102 Study Notes Jennifer Williams How Telomerase works:  Telomerase (Enzyme) prevents chromosome shortening by attaching many copies of repeated DNA sequences to the ends of chromosomes - provides upstream site for RNA primer  Step 1: Telomerase binds to DNA repeat  Step 2: Telomerase synthesizes a 6-nucleotide repeat sequence  Step 3: Telomerase moves 6 nucleotides to the right and begins to make another repeat  Step 4: Primase makes an RNA primer near the end of the telomere, and DNA polymerase synthesizes a complementary strand that is sealed by ligase - bind phosphodiester bonds on complementary and primary strands Telomeres and aging  Body cells have a predetermined life span  Skin sample grown in a dish will double a finite number of times - infants, about 80 times; older person, about 10 to 20 times  Senescent cells have lost the capacity to divide with time( telomeres are not perfect!) Telomeres and cancer  When cells become cancerous, they divide uncontrollably  In 90% of all types of human cancers, telomerase is found at high levels  Prevents telomere shortening and may play a role in continued growth of cancer cells Biology 102 Study Notes Jennifer Williams Chapter 11: Gene Expression at the Molecular Level Central Dogma of Molecular Biology (genes specify protein structure) DNA=>*transcription*=>RNA=>*translation (using messenger RNA)*=>Protein Topics:  Errors of metabolism  The genetic code (don't need to memorize)  Transcription (DNA to RNA)  Translation (RNA to Protein) Scientists Experiment & Discovery Archibald Garrod Inborn Errors of Metabolism - Found in Alkaptonuria ("black urine") (1908)  Homogentisic acid in the urine oxidizes and turns dark brown  Garrod proposed that a lost or damaged enzyme was responsible for this Beedle and Tatum "One gene -one enzyme" concept  Experimented with Neurospora: bread mold; several advantages including: it grows quickly, normally grows on 'minimal medium', haploid, so mutations and crosses reveal effects  UV irradiation produced mutants that needed argenine to grow - was an enzyme missing?  Analysis of metabolic mutants revealed multiple enzymes that lead to argenine synthesis in a serial manner  Different stages of loss of function could be identifies, each associated with only one enzyme  As a result, Beedle and Tatum proposed that 1 gene coded for 1 unique enzyme. Thus, one gene-one enzyme hypothesis (now the one gene-one protein hypothesis... an enzyme is a protein) Transcription and Translation in Eukaryotes  Transcription, the production of mRNA, occurs in the nucleus of eukaryotes  Translation, the production of protein, occurs in the cytoplasm of eukaryotes Transcription and Translation in Prokaryotes  Transcription and Translation both occur in the cytoplasm of procaryotes  So prokaryotes can produce protein very quickly in response to the need for that protein; provides a mechanism to explain the very fast growth of prokaryotic cells  Can easily adapt to the environment; perhaps better at adapting than eukaryotes (more advanced)  Cyanobacterial akinete are organisms that originally produced oxygen in atmosphere **Movie: "Processing of Gene Information: Prokaryotes vs. Eukaryotes": http://highered.mcgraw- Transcription  Making mRNA from a DNA template  Similar to DNA replication Biology 102 Study Notes Jennifer Williams  As in replication, in transcription, addition of a new nucleotide occurs at the 3' end!  Pairing is just as in DNA H-bonding: A-U: 2 H bonds; G-C: 3 H bonds  RNA is made so that its sequence is complementary to the DNA: A transcribes U G transcribes C T transcribes A C transcribes G  Three stages of transcription  1) Initiation: The promoter functions as a recognition site for sigma factor. RNA polymerase is bound to sigma factor which causes it to recognize the promoter. Following binding, the DNA is unwound into a bubble known as the open complex  2) Elongation/synthesis of the RNA transcript: Sigma factor is released and RNA polymerase slides along the DNA in an open complex to synthesize RNA  3) Termination: When RNA polymerase reaches the terminator, it and the RNA transcript dissociate from the DNA  Uses DNA as a template - has signals for where transcription begins  Sigma factor - identifies promoter region on DNA  Nucleotide triphosphates are added to the growing strand at the 3' end  Phosphodiester bonds are made by DNA dependent RNA polymerase  Template strand is non-coding strand; other is coding strand  Note the anti-parallel, complementary strands - both DNA strands can be coding. Langage of Genes  4 Nucleotides in DNA (4 for RNA with one change: Uracil instead of Thymine)  20 known natural amino acids had to be accounted for  If one base per amino acid, then 4 amino acids could be coded (4 )  If two bases per amino acid, then 16 amino acids could be coded (4 ) 3  If three bases per amino acid, then 64 amino acids could be coded (4 )  Minimum number for 20 would be to have three bases per amino acid; redundancy in the code. (ex. Argenine uses 6 coding options)  Conclusion: There must be a triplet code system  The Coding Unit is called a codon  Start codon = Meth (AUG)  Stop codons = UAA, UAG, UGA Flow of Coding Information  Central Dogma: DNA --> RNA --> Protein  Proposed by Frances Crick  String of amino acids has a direction relationship to nucleotide bases in RNA and DNA Biology 102 Study Notes Jennifer Williams What is a Gene?  Gene (modern definition): a gene is a nucleotide sequence that carries the information needed to produce a specific RNA or protein product Introns (Only in Eukaryotes)  Non-coding regions  Larger organism usually means more introns  Evolution of complex organisms  Walter Gilbert (early 1980s): exons code for particular protein regions and functions:  Different exons could be mixed to form proteins with different domains  Allows for rapid evolution of proteins  Introns may contain mutation in structure, but are not expressed  "Evolution by exon shuffling" Eukaryotic RNA Processing (Modifications that occur at the ends of pre-mRNA - post-transcriptional modification, which produces the final mRNA, which enters the cytoplasm for translation)  Introns are removed (Exons = "expressed")  The 5' end of the new RNA transcript is capped with a 7-methylguanosine (5' cap)  Helps in recognizing that this structure is an mRNA for translation to begin  A poly-Adenine tail is added to the 3' end  Helps to stabilize the mRNA: 100-200 adenine nucleotides are added  The transcript is transported out of the nucleus to the cytoplasm for the translation  Transcript is now in a functional state for translation to occur Spliceosome (Assigned Reading - p. 247)  1) First 2 subunits bind to 5' splice site and branch site  2) Additional subunits bind, creating a loop  3) 5' splice site is cut, 5' end of intron is connected to the branch site. Two subunits are released  4) 2' splice site is cut. Exon 1 is connected to exon 2. The intron (in the form of a loop) is released along with the rest of the subunits and is degraded.  snRNPs - small nuclear RNA and set of proteins ("SNURPS") Translation  The production of protein from a mRNA template  Ribosomes - the site of protein synthesis:  tRNA (transfer) and mRNA(messenger)  The peptidyl transferase activity (the enzymatic activity that forms the peptide bond) is carried out by the rRNA (ribosomal) - rRNA is a gene product and is not encoding a protein  Steps in Translation:  1) "Activation" of the tRNAs: Formation of the aminoacyl-tRNA (Putting amino acids onto tRNA - complementary to the anti-codon template on the tRNA molecule)  2) Imitation of the translation process: starts process of protein production  3) Elongation: the continued addition of amino acids to the growing polypeptide chain  4) Termination: the end of translation; release of the protein **Movie: "Protein Synthesis": Biology 102 Study Notes Jennifer Williams tRNA  One tRNA for each amino acid  Amino acids are attached covalently to the 3' end of the activated tRNA  The tRNAs carry the amino acids to the ribosome  Each tRNA has an anticodon of 3 bases that pair with the codons of the mRNA Ribosomes  Ribosomes 2 parts: Small & Large subunits  mRNA passes through groove in ribosome  A site receives new aminoacyl tRNA  P site receives polypeptide-bearing tRNA after peptide bond formation  E site tRNA exit ribosome Biology 102 Study Notes Jennifer Williams Chapter 12: Gene Regulation Gene Regulation  Constitutive genes are genes that are constantly transcribed  Their products always available in the cell (ex. glycolytic enzymes)  Note all genes are needed all the time, so many genes are regulated (turned on or off depending on the needs of the cell) The LAC OPERON: François Jacob & Jacques Manod (1961): discovered the regulation of genes using the lac operon in the bacterium, Escherichia coli  Lactose Metabolism in E. coli: Lac --> Glu + Gal (using B-galactosidase)  Then Gal is converted to Glu for glycolysis  When grown ion glucose: little B-galactosidase  When grown on lactose: much B-galactosidase  Also, an increase in Galactose permease, Galactoside transacetylas  Structural genes: enzyme-encoding genes  Operon: a gene complex in which structural genes are linked physically on the same DNA with controlling DNA sequences  Jacob and Manod described the first operon: the lac operon, as a gene system that regulates lactose metabolism  Repressor Action: When lactose is absent from the environment allolactose is not made and the lac repressor is free to bind to the lac operator, inhibiting the transcription of the operon. The lac repressor binds to the operator and inhibits transcription. When lactose is present the binding of allolactose to the lac repressor prevents it from binding to the lac operator site (lacP site). This permits the transcription of the lac operon. The binding of allolactose causes the conformational change that prevents the lac repressor from binding to the operator site.  LacZ: B-galactosidase, LacY: Galactose permease, LacA-Galactoside transacetylase Conversion of Lactose to Allolactose  Lactose present, a small amount of isomerizes to allolactose, an isomer of lactose  Allolactose binds the repressor protein, that then no longer binds the operator  RNA polymerase is free to transcribe the three genes of the lac operon **trp Operon is also negative control: see pages 265-267 Negative and Positive Regulators in the Body:  The lac operon: Negative control (repressor decreases gene expression)  Positive Control: activator increases gene expression  CAP: Catabolite gene Activator Protein  As glucose concentration decreases by metabolic activity, concentration of cyclic AMP (cAMP) increases. When cAMP binds to CAP; active form. CAP binds to DNA, which increases the transcription of the lac operon Eukaryotic structural genes - 3) features found in most promoters  1) Transcriptional start site  Where transcription begins  With TATA box forms core promoter: By itself results in low level basal transcription Biology 102 Study Notes Jennifer Williams  2) TATA box  Determines precise starting point for transcription  3) Response elements (aka. Regulatory elements)  DNA sequences that regulate genes. Bind regulatory proteins that control transcription at core promoter  Recognized by regulatory proteins that control initiation of transcription  Commonly at -50 to -300 base pairs upstream of the transcriptional start site  Enhancers and Silencers Pre-initiation Complex  Assembled GTFs and RNA polymerase at the TATA box  Form basal transcription apparatus  Contains: 1) RNA polymerase 2) 5 different general transcription factors (GTFs) 3) GTFs and RNA polymerase must come together at core promoter  Mediator: composed of several proteins  Partially wraps around GTFs and RNA polymerase  Mediates interaction with activators or repressors of gene activity  Controls rate at which RNA polymerase can begin transcription Activators & Repressors - Pre-initiation Complex  Activators bind to DNA: increase in gene activity  Repressors bind to DNA: decrease in gene activity  Both regulate rate of transcription  Most do not bind directly to RNA polymerase Controlling RNA Polymerase (2 ways)  1) Activators to bind to and then influence function of GTS  Improve ability of a GTF called Transcription Factor IID to initiative transcription  Repressors can inhibit function of TFIID  How it works:  Activator protein binds to Response Element  The activator protein enhances the ability of a GTF called TFIID to bind to the TATA box  TFIID promotes the assembly of the pre-initiation complex  2) Through a mediator  Activators stimulate the function of mediator by allowing faster initiation  Repressors inhibit the mediator so RNA polymerase cannot progress to elongation  How it works:  Mediator binds to the pre-initiation complex, but transcriptional initiation does not occur  An activator binds to a distant enhancer. A bend in the DNA allows the bound activator to interact with mediator. This interaction causes RNA polymerase to proceed to the elongation stage of transcription  RNA polymerase begins to transcribe an RNA molecule  3) Recruit proteins that influence DNA packing (explained in "Gene accessibility")  The transcriptional activator recruits histone acetyltransferase and ATP dependent chromatin remodelling enzymes to the region, which makes it less compact  Region is now accessible to other transcription factors and RNA polymerase II. Biology 102 Study Notes Jennifer Williams Gene accessibility  DNA is associated with proteins to form compact chromatin - closed conformation  Chromatin packing affects gene expression  Transcription is difficult or impossible in the tightly packed chromatin in the closed conformation  Access to the DNA is allowed in the loosely packed open conformation  Some activators diminish DNA compaction near a gene  Recruit proteins to loosed DNA compaction  Histone acetyltransferase attached acetyl groups to histone proteins so they don't bind DNA as tightly  ATP-dependent chromatin remodeling enzymes also loose DNA compaction Example: Steroid hormone  Transcription factor that responds to steroid hormones (Can work at low concentrations)  Steroid receptor  Hormone is an example of a small effecter molecule  Steroid hormones made by endocrine glands and secreted into bloodstream ( ex. pituitary gland)  Different cells respond to the hormone in different ways Glucocorticoid (targets many body tissues)  Can increase transcription of specific genes  Hormone released into bloodstream after meals  Transported into cells by a transporter protein and binds to glucocorticoid receptors  This binding releases proteins called chaperones and exposes nuclear localization signal (NLS) on the receptors  Directs the receptor to travel into the nucleus through a nuclear pore  Two glucocorticoid receptors form a dimer and travel through nuclear pores into nucleus  Dimer binds to two adjacent glucocorticoid response elements (GREs) next to particular genes (bind to different sites within different tissues)  GREs are enhancers  Increased transcription of the adjacent gene  Example: Liver = causes secretion of glucose into bloodstream Transcription Factor Motifs  Transcription factor proteins contain domains with specific function  Motif - domains or portions of domains with similar structures in different proteins  Alpha helix - important in recognition of DNA double helix  Zinc fingers can recognize DNA sequences within major groove Translational Control  One example: Mechanism of action of micro RNA (miRNA)  Step 1: The double stranded region of the pre-miRNA or pre-siRNA s cut by dicer and releases a 22 base pair RNA  Step 2: A single stranded miRNA or siRNA associates with proteins to form RISC (RNA- induced silencing omplex)  Step 3: RISC binds to a cellular mRNA due to complementarity with the miRNA or siRNA within RISC Biology 102 Study Notes Jennifer Williams  Step 4: The mRNA is degraded (high complementarity) or translation is inhibited (low complementarity)  MicroDNA as Drugs (video)  Drugs designed to block microRNA to switch off genes  Most microRNA are unkown for function, but those that we know, we have an understanding microRNA impacts disease (one for decreasing cholesterol levels) DNA methylation (another way to regulate genes)  DNA methylase attaches methyl groups -CH to3C  Common in some eukaryotes (not all)  IN mammals, 5% of DNA is methylated  Usually inhibits transcription  CpG islands found near promoters in vertebrates and plants  C and G with their phosphodiester bonds  unmethylated CpG islands = active genes  methylated CpG islands = repressed genes  Methylation can inhibit expression in 2 ways....  1) Prevents activators binding to Response elements and Enhancers  2) Converts chromatin from open to closed conformation  Methyl-CpG-binding proteins recruit proteins that condense the chromatin  See page 281 Multiple Genes Compensate for Dosages Problems  1 gene may not be enough; need multiple copies of genes  Tandemly repeated gene sequences occur one after another on chromosomes  Also selective gene replication in some cells  Gene Amplification  Gene Amplification in Tumours: Her2 in tumours of breast cancer Eukaryotic Gene Regulation  Complex Regulation: genes encode single proteins  Gene expression during certain conditions  Also Developmental, Temporal, and Tissue-specific Control of Development  Homeotic Genes: control the formation of specific structures during development (Text: see pages 418- 420)  Homeotic mutations can result in one set of cells encoding for an entirely different set Homeotic Genes  Homeotic genes contain 180 base pair sequences called homeoboxes  Homeoboxes code a protein region called a homeodomain of 60 amino acids that form four helices, one of which binds DNA and affect transcription  Homeotic genes such as Antennapedia and bithorax are often clustered as hox genes  The order of arrangement of hox genes reflects spatial organization of the animal and the order of activation during development  Are related in mouse and fruit flies  Hox genes are seen in many organisms, both segmented and non-segmented Biology 102 Study Notes Jennifer Williams Two Aspects of the Gene (Movie)  Species of flies that have a black spot on their wings is used to attract females through a courtship dance  Genes can be turned on by the organism Port Transcriptional Control  Eukaryotic RNA is protected by the 3' poly-A tail and 5' G-cap  Some eukaryotic RNA is differentially degraded  Translational controls:  Regulated translation of mRNA into protein  Usually relate to ribosomal affinity for the mRNA Post-Translational Regulation ("Enzymes affected directly")  Feedback (end-product) inhibition  Phosphorylation  Other enzyme modifications e.g. sugars  Proteolytic processing  Selective degradation Example: Precursor proteins clipped to activate: Single chain pro insulin (86 a.a.) --> proteolytic activation --> Double chain insulin (51 a.a.) Biological Complexity and the Sizes of Genomes and Proteomes  Alternative splicing can increase the proteome size without increasing the total number of genes  For organisms to become more complex, as in higher plants and animals, evolution has produced more complex proteomes  General trend is that less complex organisms tend to have fewer genes  Frequency of alternative splicing increases with increasing biological complexity (humans have 70 of genes alternatively spliced!) Biology 102 Study Notes Jennifer Williams Chapter 13: Mutation, DNA Repair, the Cell Cycle, and Cancer Mutations  Mutation: any heritable change in genetic material (DNA)  Can be brought about by mistakes in replication  Also by chemical treatments (mutagens)  Mutagens that cause cancer = carcinogens  Essential to the continuity of life  Source of variation for natural selection (Darwin's problem)  New mutations are more likely to be harmful than beneficial  Classes of mutations  Chromosomal, Point mutations, Frameshift Chromosomal Mutations  Deletion or addition of a DNA segment  Breakage  Effects: 1 to multiple proteins lost = very serious, often lethal  Example: Fragile X Syndrome Point Mutations  Affects 1 amino acid  3 types of Base Substitutions:  1) Silent or Neutral = no effect due to redundancy in code)  i.e. AAG (lysine) to AAA (also lysine)  2) Missense = "wrong sense or wrong information"  i.e. AAG (lysine) to AAC (asparagine)  3) Nonsense = "no readout or no information"  i.e. AAG (lysine) to UAG (STOP)  Example: Sickle cell Anemia (Missense in beta-globin - Glu 6 to Val 6) Frameshift Mutations  Starting point is very important because the data in genes are read in groups of three bases (codons)  A frameshift is a shift in the reading frame away from the original alignment  Removal or addition of 1/2 bases substantially changes the output  Examples: Insertion, Deletion (See diagram below) Gene mutations outside of coding sequences  Promoter (up and down promoter mutations) Biology 102 Study Notes Jennifer Williams  DNA Transcriptional response element/operator site, in the promoter: Alters regulation of transcription  Example: Lac Operon operator  Splice junctions  Mutations at the boundaries between introns and exons can prevent proper splicing  No longer recognized as a "splice zone"  RNA Translational response elements (within mRNA and not DNA)  May prevent proper translational regulation Genetics of Hemophilia (Guest (Audio) Speaker - Dr. David Lillicrap) **PEDIGREE  X-linked disease, so males (XY) are more affected than females (XX)  Recessive on X chromosomes  Male would need only 1 X chromosome to possess the disease, while females would need 2 X chromosomes  Restriction Enzymes: Enzymes that cut specific sequences in DNA (ex. Taq1)  No longer cuts if mutation occurs  Intron Mutations = Incorrect Splicing  Single nucleotide polymorphisms (SNPs) - change at a point in the DNA  Biallelic (2 forms of alleles or gene types)  DNA Repeats - Microsatellite Repeats  Rational for Hemophilia Genetic Testing (How humans are tested for Hemophilia)  Carrier diagnosis  Prenatal testing  Prediction of treatment outcomes and complications  Generation family - using polymorphism changes; usually marked by repeated introns 13 and 22 on an X-chromosome  "A" Insertion into a run of Ademinines - frameshift  Use direct analysis of nucleotides instead of indirect  Over 700 mutations result in Hemophilia  Hemophilia Mutation Databases  Mainly point mutations in both Hemophilia A (783 mutations) and Hemophilia B (749 mutations)  Pedigree (See below, right)  Circle = female; Square = male  Filled in = infected; X through or half filled in = carrier Interesting Note: Queen Victoria was a carrier of Hemophilia B - Factor 9 (thought to be the original carrier) How do mutations occurs?  Ester & Joshua Lederberg tested this. They found that only rare mutated cells survived selection process, and so mutations are random events  Experiment: Placed individual bacterial cells onto growth medium and allowed cells to Biology 102 Study Notes Jennifer Williams divide during which time random mutations may occur  Bacteria was incubated overnight to allow the formation of bacterial colonies. This is called the master plate. Bacterial colonies included one in which some cells have random mutation that gives resistance to T1, and others in which there were no mutations  A velvet cloth wrapped over a cylinder was pressed onto the master plate, and then gently lifted to obtain a replica of each bacterial colony. The replica was pressed onto 2 secondary plates that contained T1 phage.. The bacteria then incubated overnight to grow  Colonies on each plate were found in the same locations. As a result, they pre-existed as random events before the selection was applied  Germ-line or somatic cell mutations  Timing and Location of a mutation is critical to 1) severity of effect; and 2) ability to pass on the mutations  Germ-line cells give rise to gametes  Can occur in a sperm or egg cell or in cell that gives rise to eggs and sperm  Entire body's cells are then infected with the mutation  Somatic cells are all other body cells  Can occur early or late in development  Genetic mosaic results from patches of mutated tissue  Spontaneous mutations  Approximately 1 mutations in every 1 million genes  Varies between species and between genes  Induced mutations by environmental agenda  Higher than spontaneous mutation rate  Mutagens are chemical or physical agenda (UV radiation, eaten chemicals)  X and Gamma rays  Base deletions, breaks in 1 or both DNA strands  Ultraviolet rays can cause formation of thymine dimer causing gaps or incorporation of incorrect bases Biology 102 Study Notes Jennifer Williams Common Causes of Gene Mutations Common Causes of Mutations Description Spontaneous Mutations Errors in DNA Replication A mistake by DNA polymerase may cause a point mutation Toxic metabolic products The products of normal metabolic process may be reactive chemicals such as free radicals that can alter the structure of DNA Spontaneous changes in On rare occasions, the linkage between purines and deoxyribose nucleotide structure can spontaneously break. Also, changes in base structure (isomerisation) may cause mispairing during DNA replication Transponsons Transponsons are small segments of DNA that can insert at various sites in the genome. If they inset into a gene, they may inactivate the gene Induced Mutations Chemical agents Chemical substances, such as benzo(a)-pyrene, a chemical found in cigarette smoke, may cause changes in the structure of DNA Physical agenda Physical agents such as UV (ultraviolet) light and X-rays can damage the DNA. Gamma rays too. Ames Test  Bruce Ames developed a test for determining if a chemical is a mutagen  Used Salmonella typhimurium strain that cannot synthesize histidine due to a point mutation  Bacteria needs histidine or mutation occurs allowing synthesis of histidine  Test: monitors rate at which reverting mutation occurs (see below for process)
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