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BIOL 205
William George Bendena

Biology 205: Mendelian and Molecular Genetics 11/26/2013 9:27:00 PM Passage of Genetics  Diversity of Structure o There must be information for the differences in cellular structure between organisms. Information concerning development must also be included  Ability to Replicate o There must be some mechanism for replication so information can be passed from generation to generation  Mutability o Information must be able to change. So all members of a species are not identical and so evolution may occur  Translation o DNA is not enough to sustain an organism. You need machinery proteins to read and translate the info and build the structure o 11/26/2013 9:27:00 PM 11/26/2013 9:27:00 PM 11/26/2013 9:27:00 PM 11/26/2013 9:27:00 PM Pathways and Gene Interactions Week Six 11/26/2013 9:27:00 PM Lethal Alleles  Lethal genes are essential genes since only affected genes that impair essential functions result in death.  In diploids: Alive (+/m), Lethal (m/m) o Heterozygotes can maintain one lethal allele  In haploids: temperature sensitive alleles are useful o Wildtype at permissive temperature o Mutant at restrictive temperature o Missense mutation may lead to temperature sensitive phenotype Pathways  Biosynthetic Pathways o Precursor 1  precursor 2  product o Each step catalyzed by a different enzyme  Regulatory Pathways o Signal-transduction Pathways: instructions are transmitted from an extracellular signal such as a hormone. This signal activated a cellular protein that activated another and so on. This cascade of activation generally ends in the activation or inactivation of transcription of genes. o Developmental Pathways: diverse set of processes, many controlled by signal transduction that promote growth and differentiation of the body  When synthetic pathways are disturbed they result in disease Gene Interactions  General Strategy of Geneticists o Treat cells/organism with mutagen, random mutations in genes produced. Each mutant is checked to confirm single- gene inheritance. Phenotypic effect is assessed. o Test to determine the number of genes taking part and which mutations are alleles of the same gene o Form mutant crosses and observe progeny  Complementation Test o Determines if the phenotype is a result of one or multiple genes  Complementation: one allele affected on each gene (wildtype)  No Complementation: different genes (mutant) o *potential random crossover can result in heterozygous  Penetrance: % of individuals with a given allele who exhibit the phenotype  Expressivity: degree to which a given allele is expressed at phenotypic level o Intensity of phenotype. DNA Structure and Replication Week Seven 11/26/2013 9:27:00 PM Micro RNAs  Short RNAs have emerged as a new means of diagnosing and treating disease o Block translation of mRNA into protein o Initiate breakdown of normal mRNA o Some regulate one or multiple genes Implantable Sensors  Direct manipulation of individual neurons (changing memories)  Light to induce normal patterns of muscle contraction o Accurately reproduce firing order o May restore limbs paralyzed by stroke or spinal cord injury Optogenetics  Technique employed in neuroscience  Combination of optics and genetics to control the activity of individual neurons  Example: o channel rhodopsin 2 (green algae)  Activated by blue light  Causes action potentials  Depolarization activated release of neurotransmitter at synapse between neurons o Hal-rhodopsin  Generates a Cl- flux when activated by yellow light  Causes hyperpolarization thus preventing action potentials Stem Cells  Use of omnipotent cells  Very controversial Disulfide Bonds  Two cyteines in close proximity will form a covalent bond o Forms disulfide bond/bridge or dicysteine bond  This bond stabilizes tertiary structure Genetic Material  Genetic Criteria: o Information: must contain the information necessary to make an entire organism o Transmission: must be passed from parent to offspring o Replication: must be copied to allow transmission o Variation: must be capable of changes to account for the known phenotypic variation in each species  Transformation o Allow the dead S cells to transform type RS o Griffith experiment  Bacteria are capable of transferring genetic material o Hershey and Chase Experiment  DNA hold genetic material as opposed to protein DNA Structure  Nucleotide form repeating units o Phosphate group, deoxyribose sugar, AGCT  Nucleotide are link covalently by phosphodiester bonds to form a strand o Phosphate connects 5’ carbon to 3’ carbon o Directionality 5’3’  Two strands link to form a double helix which bends, folds and interacts with proteins resulting in 3D chromosomes  Packing allows ~1m of DNA to fit in a nucleus o Condensed scaffold  scaffold  solenoid -> lodish (small)  Purines: a and g  Pyrimidines: CUT  Difference between thymine and uracil is a missing methyl group and has an additional hydroxyl group forming a ribose  Phosphate and sugar molecules form the backbone of the nucleic acid strand  General Features o Two stands are twisted together around a common axis o The helix is right-handed  Turns in clockwise direction o 10 bases/complete twist o two strands are antiparallel  Double Helix o Hydrogen Bonding (between complementary bases)  AT with two hydrogen bonds  CG with three hydrogen bonds o Base Stacking  Van der Waals interactions orient bases into flattened regions o Hydrophobic Interactions  Bases on the interior  Sugar and phosphate residues hydrophilic so on the exterior interacting o Cations (Mg2+) and proteins can neutralize negative phosphate backbone Chargaff’s Rules  [T + C] = [A + G]  [T] = [A]; [C] = [G]  [A + T] does not always equal [G + C]  with two pyrimidines, DNA is too thin  with two purines, DNA is too thick DNA Replication  Semiconservative Replication o Actually right o One strand new DNA, one strand old DNA  Conservative Replication o All new or all old DNA  Dispersive Replication o Random Replication Fork  Leading Strand o Grown in the direction of the unwinding DNA fork  Lagging Strand o Requires RNA primer for DNA polymerase to start  Primase synthesizes short RNA oligonucleotides (primer)  DNA polymerase III elongates RNA primers with new DNA  DNA polymerase I removes RNA at 5’ end of neighbouring fragment and fills gap  Primer degraded by 5’3’ exonuclease activity  DNA ligase connects adjacent fragments in a process called ligation DNA Replication and Telomeres Week Eight 11/26/2013 9:27:00 PM DNA Synthesis in E. coli  General o Fast and accurate o 2000 nucleotides/second (in E.coli, two forks move at 1000 n/s) o E. coli genome is 5 million base pairs (~40) o Only one nucleotide error in 10^10 inserted (thanks to exonuclease proofreading Pol I and III)  Initiation of Replication o Origin of replication in E. coli is termed oriC (origin of chromosomal replication o Three types of DNA sequences in oriC are functionally significant:  AT-rich region  DnaA boxes: TGTGGATAA, TTATACACA, TTTGGATAA, TTATCCACA  GATC methylation sites o Many proteins are required o Unwinding the double helix  Two enzymes open the helix and prevent overwinding  Helicase (with topoisomerase is DNA gyrase): clamps to DNA, breaks hydrogen bonds ahead of DNA synthesis  composed of six subunits; 5’3’; uses ATP  SSBPs (single stranded binding proteins): bind to unwound DNA and prevent it from re-forming o DnaA proteins bind to the DnaA box sequences, the cooperative binding of an additional 20-40 DnaA proteins to form a large complex is stimulated o Replisome replicates o DNA gyrase removes extra twists and relazes DNA DNA Replication Complexes  DNA helicase and primase are physically bound to each other to form the primosome complex o Leads the way at the replication fork  Primosome is physically associated with two DNA polymerase haloenzyme complexes and associated proteins forming the replisome  Replisome o 900kd, 13 proteins, 4 subcomplexes o 2 copies of catalytic core o must copy parental DNA strands, disassemble nucleosome in parental and reassemble in daughter molecules  CAF-1: Chromatin assembly factor 1  PCNH: Proliferating Cell Nuclear Anitgen o Contains  DNA polymerase activity  3’5’ proofreading exonuclease  subunit that stimulates exonuclease  2 copies of dimerization subunit: links catalytic cores together  2 copies of CLAMP homodimer protein: binds around DNA holds catalytic core to their template  multi-protein CLAMP LOADER: places clamp on DNA o Other Information  Clamp on lagging strand dissociates at end of each okazaki fragment and reassembles at each next fragment  Leading strand is “processive”  In eukaryotes, DNA synthesis takes place during S phase of cell cycle, this is also the period of histone biosynthesis Risks  DNA double-stranded breaks (DSBs) pose the greatest challenge to cells since they result in genomic instability and high mutation frequency  Broken chromosomes can arise by insertion of a single-stranded DNA nick o Any agent that blocks topoisomerase can result in a ssNick Replication in Humans  Proceeds in two directions  Begins at three origins  Cell cycle protein cdc6at the origin of replication o Origins of replication are not evenly distributed o Gene rich regions (euchromatin) appears to be replicated first followed by heterochromatin later in the S phase.  When last primer is removed on the lagging strand, there is a terminal gap so a telomere is added Telomeres  Telomeres contain tandem repeats of short DNA sequences 10-15kb in length  Stabilize chromosomes by preventing loss of genomic info  Telomeres associate with proteins to protect chromosome ends from the cell’s DNA repair machinery o Might see ends as DSBs  Lengthening o Telomerase contains RNA (AACCCCAAC) that anneals to 3’ DNA overhang o Polymerase activity of polymerase elongates o RNA moves along DNA so that 3’ end is extended further: translocation o Polymerase activity elongates o Complementary strand is replicated  Primer synthesized by primase  Polymerase fills the gap  Primer is removed and ligase seals the gap  Werner Syndrome o Causes premature aging o Caused by mutations in the gene encoding RecQ helicase WRN (part of telomere) o Marked by aging and genomic instability (also elevated incidence of cancer)  *cancer cells often have increased telomerase activity  Mutation, Repair and Recombination Week Eight Cont. 11/26/2013 9:27:00 PM Mutation and Recombination  Two major processes responsible for genetic variation  Mutation is the ultimate course of evolutionary change o Some spontaneous, others environmental  Recombination groups new alleles into new combinations on the chromosome  Gene mutations o Point Mutation  change in a single base pair ( involves base substitution)  cannot be seen on northern blot  missense mutations cannot be seen on western blot  nonsense mutations result in smaller polypeptides making the band lower on the western blot  frameshift mutation can either shorten or lengthen polypeptide resulting in unpredictable but observable western blot migration  Types:  Transition: change of a pyrimidine to another pyrimidine or a purine to another purine (most common)  Transversion: a change of a pyrimidine >purine  Energetically unfavourable  Transversion/Transition result in:  Silent Mutations: specifies same amino acid (does not change phenotype)  Conservative Missense Mutation: altered codon specifies chemically similar amino acid  Neoconservative Missense Mutation: chemically dissimilar amino acid  Nonsense Mutation: results in stop codon  Base Insertion: results in frameshift mutation and completely different amino acid sequence  Base Deletions  Regulatory Regions o Flank the structural gene and bind to repressor proteins o Regulatory region mutations result in no mRNA or protein o Example: Lac operon is transcribed only in the presence of lactase  Binds to repressor and changes conformation Consequences of Mutations  Mutations can also occur outside of coding sequences o May alter the regulatory sequences of a gene o Change ability of DNA binding proteins to function  Eliminates transcription  Change rate of transcription  Change amount of transcription  Change the developmental timing of transcription  Eliminate transcription in response to environmental change Causes of Spontaneous Mutations  Tautomeric Shift: migration of a hydrogen atom or a proton accompanied by a switch of a single bond and adjacent double bond. Keto (GT) and Amino (AC) standard forms are rearranged into rare enol and imino forms  Depurination (most common): loss of purine base in intact nucleotide produced an apurinic site. Replacement of base may result in transversion.  Deamination: removal of amine group o C  U by loss of NH2, pairs w/A o A  hypoxanthine by loss of NH2, pairs w/C  Oxydation: formation of oxygen radicals. Addition of OH- groups to T blocks DNA replication. Added =O on G mispairs with A leads to GT transversion  Trinucelotide Repeats: duplications of normally occurring short repeated sequences o Huntington Disease: repeat in CAG in protein known as Huntington o Kennedy Disease: repeat in CAG; causes progressive muscle weakness (expansion in androgen receptor coding region) results in poly_____ o Fragile X Chromosome  A CGC repeat expansion in FMR-1 gene  Leads to mental retardation  Associated with autism  Hinders transcription (due to hypermethylation of DNA) o Slipped Mispairing  Part of the template is repeated twice in daughter strand  Mutant HTT o Blocks neurotransmitter release and stimulated apoptosis o Binds to transcription factors reducing the amount of RNA made leading to programmed neuronal cell death. Mutagenicity and DNA Repair Week Nine Mechanisms of Mutagens  Incorporation of base analogs o Chemicals that resemble normal nitrogen bases are incorporated into DNA  5-bromouracil (5-BU): analog of thymine (after ionization)  2-aminopurine: only mispairs when protonated, causes ATCG transition  Specific mispairing o Alkylating agents alter bases o EMS: ethyl group added to form ethylmethanesulfonate o NG: methyl group added to form nitrosoguanidine  Intercalating agents o Slips between bases causing insertion or deletion of a single nucleotide pair o Proflavin, acridine orange or ICR-191 can be inserted between bases  Base damage o Breaks bond between NB and DNA backbone o Results in base pair substitutions and/or replication blocks o Example: aflatoxin B1 forms bulky addition product, deadly toxin, produced by fungus Physical Mutagens  Ionizing Radiation o Includes x-rays and gamma rays o Short wave length and high energy o Can penetrate deeply into biological molecules o Creates chemically reactive molecules termed free radicals o Can cause:  Base deletions  Single nicks in DNA strands  Cross-linking  Chromosomal breaks  Nonionizing Radiation o Includes UV light o Has less energy o Cannot penetrate deeply into molecules o Causes formation of cross-linked thymine dimers and 6-4 photoproducts  They may cause mutations when DNA strand is replicated Ames Test  Evaluates mutagenicity  Developed by Bruce Ames  Uses a strain of Salmonella typhimurium that cannot synthesize the amino acid histidine o Due to point mutation in a gene involved in histidine biosynthesis  A second mutation (reversion) may occur restoring the ability to synthesize histidine  Monitors rate at which this second mutation occurs  Some salmonella strains revert through base-pair substitution, others require frameshift DNA Repair  Since most mutations are deletions, DNA repair systems are vital to survival of all organisms  In most cases, DNA repair is a multi-step process o An irregularity in DNA structure is detected o The abnormal DNA is removed o Normal DNA is synthesize  Proofreading function of DNA polymerase I and III first defenses  Common types of DNA repair systems: o Direct Repair  an enzyme recognizes an incorrect alteration in DNA structure and directly converts it back to its correct structure  enzymes involved:  Photolyase  Repairs thymine dimer by splitting them  O6- alkylguanine alkyltransferase  Repairs alkylated bases by transferring a methyl or ethyl group from the base to a cysteine side chain within the alkyltransferase protein (permanently inactivating alkyltransferase)  CPD photolyase  Can repair photodimers o Base Excision and Nucleotide Excision Repair  an abnormal base or nucleotide is first recognized and removed from the DNA. A segment of DNA in this region is excised and then the complementary DNA strand is used as a template to synthesize normal DNA  Syndromes due to defects in NER:  Xeroduma Pigmentosum  UV light sensitivity, skin cancer  Cockayne Syndrome  UV light sensitivity  Developmental disorders o Dwarfism, mental retardation, deafness, premature aging…  NER normally activated when replication fork stalls or is blocked  Pathways:  Global Genomic Repair (GGR)  Corrects anywhere in the genome  Recognizes damaged base by heterodimeric complex  Damaged DNA, with complex proteins bound, recruits general transcription factor (TF IIH) (10 proteins)  XPB and XPD helicases mutated in Xeroduma Pigmentosum  Assembly of multiprotein complex and unwinding o RPA (SSBP) stabilizes structure  Incision of damaged DNA o One 15-24 nucelotides from lesion o Other (closer) 2-8 nucleotides  Synthesis and ligation of DNA o PCNA (sliding clamp), bypass polymerase, ligase 1  Transcription- Coupled NER  Repairs transcribed regions  Recognition of stalled transcription complex by CSA/CSB (mutated in Cockayne Syndrome)  XPB and XPD helicases mutated  Mismatch Repair o Similar to excision repair except that the DNA defect is a base pair mismatch in the SNA, not an abnormal nucleotide. The mismatch is recognized and a segment of DNA in this region is removed. The parental strand is used to synthesize a normal daughter strand. o Repair of mismatches missed by the 3’5’ exonuclease of DNA polymerase III o Lack of this repair system associated with some hereditary forms of colon cancers o Corrects replicative errors o Steps:  Mispairing creates a distortion  MutS recognizaes mismatched pair  MutH recognizes methylated parent strand and nick daughter strand  MutH nicks the GATC methylation site on the daughter strand A that is not methylated  UVRa (a helicase) binds to nick and unwinds DNA  New strand excised and replaced between nick and mismatched pair  Proteins bind to parental ssDNA to protect  Daughter strand between nick and lesion excised  Recombinational Repair o occurs when DNA damage causes a gap in synthesis during DNA replication. The gap is exchanged between the abnormal DNA and the corresponding region in the normal, replicated double helix. After this occurs, it is possible to fill in the gap using the complementary DNA strand.  SOS Mechanism o Polymerase III stalls at sight of damage o Polymerase IV  replaces polymerase III (signaled by Rec A protein)  Larger active site  Error prone (no proofreading 3’5’ nuclease)  Only adds a few nucleotides before falling off o Addition of a single ubiquitin molecule to the sliding clamp or PCNA (proliferating cell nuclear antigen) changes conformation so that it binds polymerase V  Rad 6 and Rad 18 (ubiquitin ligase) catalyzes Ub addition  Example of regulation by post-transcriptional modification Double Stranded Breaks  Complementary cannot be exploited (nonhomologous)  KU*) and KU70 proteins recognize two ends, bring them together and ligate using XRCC4 and DNA ligase IV  Error-free repair by SDSA (synthesis dependent stand annealing) o Homologous o Uses sister chromatids available in mitosis as templates o Proteins bind to broken ends o 5’ends are trimmed by an endonuclease to expose a SS region o RecA/Rad51 form long filaments with ssDNA  Searches/invades sister chromatid for homologous sequence  Displacement (D) loop caused by invasion of 3’ end Cancerous Cells  Rapid division and metabolic rate  Ability to invade new cellular territories  Most common being mutation in oncogenes and tumor suppressor genes  Oncogenes o Gain of function dominant mutations o Mutation needs to only be in one allele o In non-mutated form known as proto-oncogenes o Proteins encoded by oncogenes are usually activated in tumor cells  Tumor Suppressor Genes o Encode proteins whose loss of activity can contribute to a cancerous state o Loss of function (recessive mutations) o For cancer to develop the mutation must be in both alleles  Eg. BRCA1 and BRCA2 Wild-type Protein Function Gene Type Promotes cell-cycle progression Oncogene (gain of function) Inhibits cell-cycle progression Tumor-Suppressor Gene (loss of function) Promotes apoptosis Tumor-Suppressor Gene (loss of function) Inhibits apoptosis Oncogene (gain of function)  Protooncogenes o Proteins that induce (positively control) the cell cycle:  Growth factor receptors  Signal transduction proteins  Transcription regulators o Other proto-oncogenes inhibit (negatively control) the apoptotic pathway  Ras oncogene is always active; fueled by GTP Chromosome Numerical Variations  Polyploidy o Extra complete set(s) of chromosome(s) o Found in some spontaneously aborted human fetuses  Aneuploidy o Wrong number of chromosomes o Examples  Trisomy: 2n+1; the presence of an extra copy of one specific chromosome  Monosomy: 2n-1 o Origins of aneuploidy most frequently lie in meiotic non- disjunction o Common Aneuplodies  Turner Syndrome (XO, monosomy)  Brown spots  Folds of skin  Poorly developed breasts  No menstruation  Short stature  Rudimentary ovaries  Klinefelter Syndrome (XXY) *XXX and XYY also viable  Male  Osteoporosis  Small testes  Talls tature  Mildly impaired intelligence  Breast development (30%)  Down’s Syndrome (Ts21)  Extra 21 chromosome  Incidence of non-disjunction increases w/maternal age  Patau Syndrome (Ts13)  Edwards Syndrom (Ts18) o Abnormalities and lethality associated with monosomy and trisomy suggest light dosage control with no dosage compensation o Non-disjunction at first division leads to all aneuploidy products o Non-disjunction at second division leads to 2 normal, 2 abormal Structural Aberrations  Most frequent include deletions, inversion, duplications and translocations  Abnormalities in structure can lead to abnormalities in meiotic segregation of chromosomes as well as further structural abnormalities after crossover  Translocations can be reciprocal or non-reciproca
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