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Biology 1001A
Tom Haffie

Lecture18 retrovirus: a virus with an RNA genome that replicates via a DNA intermediate retrotransposon: a transposable element that transposes via an intermediate RNA copy of the transposable element thymine dimer: a pair of abnormally bonded adjacent thymine bases in DNA, resulting from damage by UV radiation, the cellular processes that repair this often make errors that create mutations photolyase: DNA repair enzymes that repair damage caused by exposure to UV light excision repair: term applied to several DNA repair mechanisms, that remove damaged nucleotide replacing it with an undamaged nucleotide complementary to the nucleotide in the undamaged DNA strand radioactivity: the giving off of particles of matter and energy by decaying nuclei ionizing radiation: consists of particles or electromagnetic waves that are energetic enough to detach electrons from atoms or molecules, thus ionizing them aneuploid: an individual with extra or missing chromosomes isotope: a distinct form of the atoms of an element, with the same number of protons but a different number of neutrons nondisjunction: the failure of homologous pairs to separate during the first meiotic division or of chromatids to separate during the second meiotic division misdivision: wrong division of chromosomes, during meiosis II deletion: chromosomal alteration that occurs if a broken segment is lost from a chromosome duplication: chromosomal alteration that occurs if a segment is broken from one chromosome and inserted into its homologue inversion: chromosomal alteration that occurs if a broken segment reattaches to the same chromosome from which it was lost, but in reversed orientation, so that the order of genes in the segment is reversed with respect to the other genes of the chromosome translocation: a chromosomal alteration that occurs if a broken segment is attached to a different, non homologous chromosome mechanism of action of DNA damage by non-ionizing radiation(UV) - UV light is absorbed by DNA, photons of UV light are absorbed in particular where there are two thymines side by side, those two circular rings absorb the photon, reorganizes the DNA so that the two thymines end up covalently linked together in ways they aren’t supposed to (thymine dimer) - this causes a huge bump in the helix - the thymine dimer in chromosomes in fatal, cannot replicate through that damage - these dimers can be repaired by photolyase of excision mechanism of action of DNA repair enzymes - photolyase breaks the thymine bonds and restores the thymines back to normal - photolyase is driven by photons of light white (visible part of sunlight) because when you are exposed to UV light you are also exposed to white light - another way to repair them is to chop them out, distortion is so easily found, they make a nick on either side and replace that space with a pair of A’s as it should be (excision) - fixing this damage can result in mutation because these repair mechanisms are so error prone mechanism of action of DNA damage by ionizing mutagensis - when they pass through your tissue, ionize, and you end up with reactive oxygen species - reactive oxygen is what damages the DNA - reactive oxygen can oxidize the bases on your DNA in a way that causes them to base pair wrong, in ways that cause them to call for the wrong pairing partner - or ionizing radiation can create reactive oxygen that breaks the double helix mechanism of production of aneuploid gametes during meiosis - exposure to radiation might mess up chromosome division - effects the cytoskeleton that give rise to unbalanced gametes - exposure to radiation is mutagenic in this way - gives rise to large scale genome mutation - break chromosomes and cells try to repair this but in not perfect ways (deletion, insertion, translocation) difficulties of studying effect of radiation exposure on large populations - area of study is called epidemiology - down syndrome spiked 9 months after in Belarus - go looking for thyroid cancer, after the accident there are increases in thyroid cancer in adolescents - higher in adolescent because they were in their developing stages - but the cases are so small (4 in 100, 000) that it could have been many other things - put more money in health screening, if they had missed one person there would not have been as a significant rise Lecture19 cyclin: in eukaryotes, protein that regulates the activity of CDK (cyclin-dependent kinase) and controls progression through the cell cycle CDK: a protein kinase that controls the cell cycle in eukaryotes carcinogen: substance that produces cancer proto-oncogene: is a normal gene that can become an oncogene (gene that has the potential to cause cancer) due to mutation or increased expression tumor suppressor gene: a gene that encodes proteins that inhibit cell division risk factor: a variable associated with an increased risk of disease or infection top four most common cancers in Canada - 40% of women in Canada could get cancer (25% of dying from cancer), 45% of men could get cancer (30% of dying from cancer) - lung cancer, prostate cancer, breast cancer, colon cancer example of proto-oncogene - proto-oncogenes are good, they are essential for normal growth and development - EGFR (epidermal growth factor receptor), transmembrane protein, has active site outside cells that binds protein EGF, it stimulates cells to divide - signals the presence of EGF, outside the cell on the inside of the cell possible mechanisms of up-regulation of proto-oncogenes - EGRF detects EGF outside of the cell, signals into the cell and effects the binding of transcription factors in the nucleus - any of these parts could be carcinogenic, resulting in too much expression - proto-oncogenes can be activated and deregulated to oncogenes - some sort of mutation, that is is always signaling can cause cell to divide and divide uncontrollably - proto-oncogene can become activated by being moved to a new place, base substitution, mutation in promotor, regulation problem with repressors and enhancers, example of tumor suppressor gene - job it is to put the breaks on cell division - TP53, transcription factor that binds the promotor of many target genes - cells can undergo stress or shortening telomeres can induce tumor repressor gene - target genes effect DNA repair, G1 arrest, Apoptosis - if cells become too damaged, TP53 tells the cell to go die possible mechanisms of inactivation of tumor suppressor gene - mutation in the promotor might shut it off, get highly methylated and shuts off promotor in particular, the possible role of miRNA in cancer - miRNA mutation that mistakenly shuts off tumor repressor gene reason why certain cancers are more frequent in certain families - gene has epigenetic marks that increases the risk - activated oncogene destroys the embryo - they’re inheriting an inactivated tumor suppressor gene - so they are heterozygotes for suppressor gene but they are not inheriting cancer because they have one suppressor gene that is active and one that is inactive, if the other become inactive then that is when people get cancer main risk factor associated with cancer cause by HPV - HPV is a carcinogenic DNA virus, not a retrovirus, does not insert into your chromosomes - it destabilizes the cell cycle, causes increased growth (warts) in different tissues - some strains are oncogenic; particularly strong association of strain 16 and 18 with cervical cancer - HPV is sexually transmitted virus - first year of university you are likely to contract HPV because of increased sexual partners - risk factors: number of new partners (more than one new partner, risk goes up 10%), frequency of condom use by partner (highest risk is sometimes using one), number of previous partners of sex partners (unknown is riskiest) mechanism by which HPV causes cervical cancer - causes cells in your cervix to become abnormal and these cells can turn into cancer, cause tumors because they multiply out of control Lecture20 somatic gene therapy: gene therapy in which genes are introduced into somatic cells germ line gene therapy: experiment in which a gene is introduced into germ-line cells of an animal to correct genetic disorder electroporation: the application of an electric current to a living surface in order to open pores or channels through which something my pass biolistics: involves directly shooting a piece of DNA into the recipient plant tissue Ti plasmids: tumor-inducing plasmid, used to make transgenic plants viruses: an infectious agent that contains either DNA or RNA surrounded by a protein coat adeno-associated viruses: a small virus which infects humans and some other primate species ITR: inverted terminal repeats, job is to integrate DNA in the right spot, without them it would be random lytic infection: penetration of a host cell by a lytic phage expression vector: a plasmid that is used to introduce a specific gene into a target cell transgene: a gene that is transferred from an organism of one species to an organism of another species by genetic engineering packaging cell: modified for the production of recombinant retroviruses, they express essential viral genes that are lacking in the recombinant retroviral vector transduction: in cell signaling, the process of changing a signal into the form necessary to cause the cellular response. in prokaryotes, the process in which DNA is transferred from donor to recipient bacterial cells by an infecting bacteriophage homologous recombination: type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA genomic DNA: set of DNA that comprises the genome of an organism restriction endonuclease digest: an enzyme that cuts DNA at a specific sequence agarose gel electrophoresis: method used to separate a mixed population of DNA and RNA fragments by length to estimate the size of DNA and RNA fragments or to separate proteins by charge Southern blot: a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples Northern blot: technique used in molecular biology research to study gene expression by detection of RNA in a sample polymerase chain reaction: scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence primer: a short nucleotide chain made of RNA that is laid down as the first series of nucleotides in a new DNA strand or made of DNA for use in the polymerase chain reaction hybridization: to cause to interbreed or combine so as to produce hybrids (offspring of two animals of different breeds), somatic cells two different species, two DNAs denaturation: a loss of both structure and function of a protein due to extreme conditions that unfold it from its conformation annealing: to heat and then cool double stranded nucleic acid in order to separate strands and induce combination at lower temperatures especially with complementary strands cloning vector: is a small piece of DNA into which a foreign DNA fragment can be inserted pBluescript: a commercially available phagemid containing several useful sequences for use in cloning with bacteriophage selectable marker: a gene introduced into a cell, especially a bacterium or to cell in culture, that confers a trait suitable for artificial selection multiple cloning site: also called a polylinker, is a short segment of DNA which contains many restriction sites - a standard feature of engineered plasmids transformation: the conversion of the hereditary type of a cell by the uptake of DNA released by the breakdown of another cell X-gal: an organic compound consisting of galactose linked to a substituted indole reverse transcription PCR: an enzyme the uses RNA as a template to make a DNA copy of the retrotransposon, it is used to make DNA copies of RNA in test tube reactions transcript abundance: oligo-dT primer: complementary (cDNA): is DNA synthesized from a mature RNA template in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA polymerase DNAase1: enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone types of disorders that lend themselves to gene therapies - diseases caused by single gene defects mechanisms for transferring DNA into cells - somatic gene therapy and germ-line gene delivery - germ-line means transforming the genes in the egg or sperm - somatic means introducing the wildtype gene into a specific somatic cell - electroporation, take naked DNA and use electrical current to enter DNA - monoinjection, single cell and use needle to puncture membrane and insert DNA - biolistics, shoot DNA into cells - Ti plasmid, plant specific - viruses, infectious, inject DNA so why not use them as a vehicle to get into cells problems that arise in using viruses as gene therapy vectors - pathogenicity, will it get you sick or make you die? - immune response, you detect something foreign so the immune response is very severe, killing you - inserted randomly, the DNA just goes anywhere in the cell, not to the specific place you want structure and function of wild type and recombinant AAV - very common in human population, doesn’t cause disease and have very mild immune response and is also very specific - by itself it doesn’t do much in you cell, the AAV genome integrates at a unique site on chromosome 19 - causes lytic infection by destroying some cells and infecting others - doesn’t replicate on its own, it requires co-infection to propogate, needs another virus to provide protein, etc (herpes or adenovirus) - want to make rAAV genome (recombinant) - take the backbone of wild-type and put in promotor and transgene, let them infect packaging cell, let if grow and you are left with rAAV particles - use these to infect cells in someones body, this is called transduction - stable insertion, so immune response is minimal mechanism by which site-specific homologous recombination “switches” disease allele - two crossover events, you swap wildtype with mutation - transgene is seeing just one mutation, brought into one cell, crossing over occurs and the mutation is not on the viral DNA, problem fixed mechanism of action of restriction endonuclease - you can easily isolate genomic DNA - you can digest it by using restriction endonuclease - cut up undigested DNA so that you get different fragment lengths - run that on a gel, you get a smear usefulness of PCR - polymerase chain reaction - allows you to amplify specific sequences of DNA - you can amplify enough DNA so we can isolate it and look at it components required for PCR - double stranded DNA, very small amount of DNA you want to amplify - DNA template, Two oligonucleotide primers, deoxynucleotides, buffer components and water, thermostable DNA polymerase - PCR machine raises and lowers temperature very fast continuously mechanism of PCR - first thing you do is raise the temperature, denature it, strands will come apart (94 degrees, breaks hydrogen bonds) - lower the temperature and primers will anneal (45-55 degrees for 20-30 seconds) it will stick through hydrogen bonding to each strand, time depends on the primer - extension occurs, DNA synthesis (72 degrees) nucleotides add to 3’ end of primer - cycle takes 3 or 4 minutes to complete, 30 cycles you can amplify a billion fold formula to estimate the chance of finding any given sequence in a genome - 1/4 chance of finding an A, G, C or T - 1/16 chance of finding any dinucleotide sequence - 1/256 chance of finding a given 4-base sequence components of plasmid cloning vector - has selectable markers - amphiphile resistant gene, if it is in a bacterium it would grow in a environment with amphiphilant while all other would die - has an origin of replication so it can replicate itself - lacZ required for synthesis of B-galactosidase mechanism of getting plasmid vectors into host cells - transform them into the bacterium - and then inject it into the cell significance of blue vs white transformant colonies growing on plate containing X-gal - in presence of B-galactosidase it turns blue - if it is white, they represent bacterias that are harboring the cloned gene interpretation of patterns of restriction endonuclease digest fragments on agarose gels - can isolate plasmid DNA and run it on a gel - if you cut with either enzyme you get two of the same length - this confirms that you have the right segment of DNA mechanism of reverse transcript PCR to make cDNA - most RNA is ribosomal RNA - mRNA has a poly-A tail (it is poly-adenilated), so you can use a primer that is just a bunch of T’s - and this will extend using reverse transcriptase to make cDNA - then you can amplify the cDNA - the abundance of mRNA reflects the abundance of amplified cDNA importance of treating with DNAase1 in RT PCR - treat you RNA with DNAase1 enzyme to take out any contamination - DNAase1 will destroy any residual DNA - this will ensure you are amplifying cDNA Lecture21 GMO (genetically modified organism): an organism whose genetic material has been altered using genetic engineering techniques Agrobacterium: a genus that uses horizontal gene transfer to cause tumors in plants Ti plasmid vector: Ti plasmid is a circular plasmid that often, but not always, is a part of the genetic equipment that Agrobacterium use to transduce its genetic material to plants T-DNA (transfer DNA): the transferred DNA of the Ti plasmid of some species of bacteria such as Agrobacterium auxin: class of plant growth substances and morphogens, have an essential role in coordination of many growth and behavioral processes in the plant’s life cycle cytokinin: a class of plant growth substances that promote cell division, or cytokinesis, in plant roots and shoots ethylene: hormone in plants, it acts at trace levels throughout the life of the plant by stimulation or regulation the ripening of fruit, the opening of flowers, and the shedding of leaves opine: low molecular weight compounds found in plant crown gall tumors or hairy root tumors produced by parasitic bacteria of the genus Agrobacterium co-culture: growth of distinct cell types in a combined culture callus: the tissue that forms over the wounds of plants protecting the inner tissues and causing healing totipotent: having the potential for developing in various specialized ways in response to external or internal stimuli RNAi (RNA interference): is a system within living cells that takes part in controlling which genes are active and how active they are siRNA (small interfering RNA): is a class of double stranded RNA molecules, involved in RNAi pathway where it interferes with the expression of a specific gene RISC: RNA-induced silencing complex is a multi protein complex that incorporates one strand of a siRNA or miRNA, uses siRNA or miRNA as a template for recognizing complementary mRNA ACC oxidase: catalyzes the last step in the biosynthesis of the plant hormone ethylene golden rice: rice produced through genetic engineering, developed as a fortified food to be used in areas where there is a shortage of dietary vitamin A carotene: used for several related hydrocarbon substances having the formula C40Hx, which are synthesized by plants but cannot be made by animals, it is an orange photosynthetic pigment important for photosynthesis vitamin A: needed by the retina of the eye in the form of a specific metabolite, the light-absorbing molecule retinal aleurone: protein found in the protein granules of maturing seeds and tubers, also used for the outermost cell layer of the endosperm endosperm: the tissue produced inside the seeds of most flowering plants around the time of fertilization Bt corn: corn that has been genetically modified to protect it from pests eutrophication: the addition of artificial or natural substances, such as nitrate and phosphates, through fertilizers or sewage, to an aquatic system how natural mechanism of transformation of plants by Ti plasmid is used to create GMO - bacteria is in the soil, is there is a wound site on the plant the bacterium can detect that and infects the wound site (wound site releases compounds that are sensed by the bacterium - Ti plasmid, part of it gets transferred and incorporated into the genome of the injected plant cell, T-DNA is the transferred DNA that gets moved over - insertion is random, no specific location - insertion causes a tumor like growth which causes the crown gall function of “left and right border” sequences of T-DNA - sequences at either end that aid in the T-DNA being add at random into the plant genome - sections in the T-DNA that code for plant hormones - left and right border define the insertion into the plant genome usefulness of cauliflower mosaic virus 35S promoter in expression vectors - a pieces of DNA that leads to very high and constitutive expression of whatever the gene is behind it - leads to very high levels of transcription - we build this in because we always want this gene to be on, we don’t want a promotor that only turns on in certain instances use of siRNA transgenes to inhibit expression of selected genes - RNA interference, umbrella term that includes the formation of siRNA - based on double stranded RNA, dicer crops them up into siRNA (still double stranded) - gets unwound to create single strand (20 nucleotides in length) one of these is antisense to a wild-type transcript - one will anneal to normal RNA, this causes translation arrest or tags the RNA to be broken down, either way the gene is silenced - you produce double stranded RNA when you put gene into plasmid - knock down the expression of ACC oxidase so that fruit doesn’t ripen too quickly using siRNA line - post-transcriptional gene silencing relationship between vitamin A deficiency and blindness - vitamin A can be synthesized from B-carotene - B-carotene to Retinol to Retinal to 11-cis Retinal - photoreceptor in your eye is 11-cis Retinal, so if you do not get enough B-carotene you do not have enough 11-cis Retinal how golden rice was constructed to express carotene in endosperm - inserted 3 genes in T-DNA to create golden rice - reduce the number of steps from GGDP to B-carotene ******* READ THE PAPER ON THIS ******** how GM plants affect agricultural practices - reduces pesticide and fertilizer use - increases the food production, crop yield - sustainable agriculture - control plant disease with genetics instead of chemistry - nitrogen from the fertilizers gets into oceans and reduces oxygen in the water and you get what is called the dead zone (lots of algae produce lots of oxygen during the day but at night, bacteria consume the oxygen until there is none left) arguments “pro” and “con” for GM plants - GM technology is unnatural: molecular breeding is really no different than traditional, more precise and faster develop new varieties (ex. teosinte, which ended up as corn after lots of crossing - GM foods are harmful to human health: no shred of evidence for this, general public has no idea what genetically altered really means - Transgenic crops threaten the ecology of natural populations: fear that the genes could escape and get into other genomes but there is no clear evidence of that happening - Transgenic crops benefit large corporations at the expense of small farmers: the large companies can patent what they’ve done and this will hurt small farmer’s incomes Lecture22 signal cascade: a series of reactions in which the products of one reaction are consumed in the next reaction hormone: any of various internally secreted compounds, as insulin or thyroxine, formed in endocrine glands, that affect the functions of specifically receptive organs or tissues when transported to them by the body fluids cytoplasmic protein domain: forward genetics: an approach that encompasses several means of identifying the gene or set of genes that are responsible for a particular phenotype within an organism ethyl methane sulfonate (EMS): a mutagenic, teratogenic and possibly carcinogenic organic compound, produces random mutations in general material by nucleotide substitution alkylating agent: used in cancer treatment that attaches an alkyl group, attached to the guanine base of DNA, at the number 7 nitrogen atom model organisms: non-human species that is extensively studied to understand particular biological phenomena, with the exception that discoveries made in the organism model will provide insight into the workings of other organisms mutagenic screen: can identify and study a phenotype of interest triple response: the three characteristic morphologic charges on an etiolated seeding in response to the presence of ethylene ETR1: ethylene binding, protein histidine kinase, two-component response regulator CTR1: constitutive triple response, always shows triple response how forward genetics techniques are useful in elucidating the steps in signal transduction pathways - signal is received by receptor, there is a signal cascade which works through intracellular membranes - hormones don’t directly effect gene expression, they bind to receptors and start the cascade - physical things like light can also start cascade - domain of the receptor becomes active after finding a signal - binding of a receptor could also switch off the pathway - the goal is to identify the genetic basis of a specific phenotype or trait by finding the mutations, but mutants are very rare and difficult to find, so make your own mutants called mutagenesis and not have to waste time looking for the mutants mechanism of mutagenesis of EMS - irradiation (UV, x-ray) - chemicals - insertional elements (transposons, T-DNA) - ethyl methyl sulfonate is a common chemical used, it introduces point mutations - EMS damages guanine, it adds a methyl group so guanine does not pair with C, it pairs with A (the G:C pair becomes A:T pair) characteristics of model organisms that make them particularly useful for genetic analysis - want a total genome size to be small so we don’t need to spend a ton of time looking through every gene - also want the life cycle to be very fast, generation time to be short so you can produce a new generation you can examine strategy for recovering homozygous mutant progeny from mutagenized Arabidopsis seed - treat seed with mutagen, should lead to a single mutation to one chromosome - plants grow and “self” - cross them with themselves, M1 is the first mutated generation - want to cross them because most defects are recessive, and only show up in homozygous recessive form - so you cross the M1s and you get 1/4 homozygous recessive form usefulness of the triple response as a screen for ethylene pathway mutants. - triple response is the screen - little seedlings, no chlorophyl in the presence of ethylene it is short, fat and has a hook at the top (this is the triple response) - in the absence of ethylene, seedlings are very tall, skinny and do not have a hook on the top different types of mutations that affect the ethylene response signal pathway - Biosynthesis: mutation in biosynthesis, in actually producing the hormone, nothing to do with signal transduction - a defect in any one of these genes would prevent ethylene from being made - may be on
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