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This 10 page Class Notes was uploaded by Lauren Nagra on Wednesday September 3, 2014. The Class Notes belongs to a course at University of California Santa Barbara taught by a professor in Fall. Since its upload, it has received 109 views.
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Date Created: 09/03/14
Monday 11252013 03092014 1105 AM CHAPTER 15 Gene Mutations and Molecular Medicine Detecting Human Genetic Variation 0 Restriction enzymes 0 O 0 DNA gel blotting PCR amplification DNA fingerprints Mutations and Human Diseases 0 O O O O O 0 Single nucleotide polymorphisms PKU Sickle Cell Trinucleotide Repeats Huntington Disease Fragile X RFLP resetriction Fragment Length Polymorphism SNP mapping of genes Treating Genetic Diseases Genetic screening Ex vivo genetic therapy direct Chronic mylogenous leukemia CML and Imatinib Gleevac Seemed like a deadly disease you would die within 5 years Translocation occurred where a piece of a chromosome from two different homologous chromosomes exchange fragments This is an ERRORmistake These happen fairly frequently in somatic cells Cancer is due to somatic mutations usually This specific one cancer happens in bone marrow that is to make white blood cells Tyrosine kinase that is no longer acting correcty this particular hybrid protein regulates whether a cell divides or not by making this the cells start dividing uncontroaby pretty much what cancer is They eventually made Gleevac Imatinib they new exactly what the target was knew what protein to inhibit went into lab and knew what to make This drug stopped cells from dividing stopped cancer Patients could now live a normal life span Bacteria fight invading virus by making restriction enzymes Restriction enzymes defend bacteria from foreign dna El Ex if viral dna comes along it can be cut by restriction enzymes The host protects itself from enzyme by putting methylation wherever it needs to but viral dna doesn39t have those so it gets caught up avery experiment 0 you can use electrophoresis to see if something cuts or not if it seperates into two bands it does if it doesn39t then it doesnT o if no cut arge frag if cut smaller spread out cuts we can use these techniques to determine genotypes for sure 0 This eliminates uncertainty 0 There are a lot of tools for dna anaylysis but we are just gonna go over the basics DNA fingerprinting 0 Two types of DNA polymorphisms are used for DNA fingerprinting Single nucleotide polymorphisms SNPs inherited variations involving a single base point mutations El biayic not that many diff snips at each base Reference genome just the most common of each base nobody actually has this genome exac y We are made of about a million snips some of them affect phenotype a lot do not Short tandem repeats STRs short repetitive sequences occurring side by side on chromosomes usually in noncoding regions El El Vary in length Highly poymorphic repeats at each site is very variable 0 1514 mom homo for 6 dad homo for2 kid has to be heterozygous in forensics we know where the repeats are and we can use that infohow 2 Anastasia example 2 you know parent inherits childs repeats 2 Anastasia was actually shot and killed as wel she was just buried somewhere else 2 They knew this because of testing the parents and children and you can use this technology to figure this stuff out see how inheritance happens Icicker father B If you look at 10 or more fragments you can pretty much prove that that person did the crime because not many people would have similar DNA probability But if 110 doesn39t match that could prove your innocence Inborn errors of metabolism Garrod Genetic mutations may make proteins dysfunctional Defects in proteins that are enzymes of metabolism can cause an inherited metabolic disease Phenylketonuria PKU results from an abronmal enzyme phnylalanine hydroxylase PAH normay catalyzes conversion of dietary phnylalanine to tyrosine You don39t need excess phenyaanine it goes to tyrosine the extra if you cannot process that it makes phenylpyruvic acid which in excess can cause mental retardation Genetic screening if we can identify these babies with these en vivo Sickle Cell Disease Hemogobin protein with quaternary structure two alpha and two beta chains In sickle cell disease one in every 146 is messed up blah blah Restriction Fragment Polymorphism RFLP One way to look for these diseases is by interrogating that snip Sickle cell is caused by a snip and the single thing that changes the amino acid is changed you can interrogate that snip with an enzyme called MST2 2 One way to do this is to make primers that flank target site flank target and add enzyme to see if it cuts in half if it does you have the restriction site if not you have the polymorphism 2 Another way aee specific oligonucleotides Short nuceotides have specific things they will align to If it is mutated anywhere along the things it will not bind You probe for each individual s DNA for normal and sickle cell 0 You can see if they are heterozygoius or homozygous 0 You don39t need restriction enzyme 0 Tuesday 11262013 03092014 1105 AM Probe will only stick if complementary sequence is matched perfectly Red signal indicates that there is dna that is perfect match Probes that have 1 pair mismatch will not stick We get signal in both spots with heterozygotes but in homozygote there is only one signal for the normal allele none for sickle allele so we know it is a homozygote for normal Polymorphisms are very common but often do not effect function they are neutral Only 3 are actually bad 0 Causing anemia other 2 not as bad as sickle cell How do we identify a disease gene in genome Complicated Locating disease genes by GENETIC LINKAGE Genetic Linkage Genetic markers such as SNPs STRs and RFLPs are used as landmarks to find genes of interest genes must be polymorphic 0 You want a marker that is inherited with disease gene To narrow the gene location a marker and gene must always be inherited together 0 If you don39t know what is wrong you just have to use linkage to find the gene When linked dna region is identified a variety of methods can identify the gene responsible for a disease Gene isolation can lead to tests for mutations protein identification and possible treatments I let39s say we have a disease gene but we don39t know what it is We do have the markers and stuff We are trying to find markers that are near by There are always markers if you look hard enough Unique locus is marked Are the markers linked How to find disease gene Do an assay of all 500 markers and see You find a few 0 You find a couple that flag your disease gene OOOOOO 0 Now that I find these I go to sequence map and see where they are and then we find candidate genes One candidate must be disease gene DNA linkage analysis 0 SNP doesn39t cause the disease we just use it to monitor because it is closely linked to the disease mutation but it does not CAUSE the disease 0 SNP that are tightly linked are very useful 0 If it wasn39t tightly linked it wouldn39t show linkage because there would be crossing over Constructing a genetic linkage map 0 Score how often certain things happen to see if marker is close to the disease gene CHcker 0 We know B and C are linked because ofA Trinucleotide Repeat Diseases 0 Expanding triplet repeats have been found in other diseases Mytonic dystrophy huntington s disease 0 How the repeats expaind is unknown possibly DNA polymerase slips after copying the triplet it copies it again Fragile X chromosome 0 Individual with this disease showed unusual X chromosomes 0 Tip would break off while making karyotype 0 Causes mental retardation in some people 0 The gene for fragie X FMR1 contains a repeated triplet CGG in the promoter region Males with a moderate number of repeats have no symptoms 0 The repeats become more numerous in successive generations In mentally retarded people with fragie X it is repeated 2002000 times 0 Genetic Screening DNA testing is direct analysis of DNA for mutation the most accurate way of detecting an abnormal allele Preimplantation screening of a zygote can be used for parents of a child with a disease like cystic fibrosis Fetal cells and newborns can be tested for sicke ce disease and others You can do abortions before once you know it has fragile X You can also make 10 embryos and see which one does not have cystic fibrosis and use that one avoids doing abortion but it39s super expensive Bioethics Should we be able to prescreen Wednesday 11272013 03092014 1105 AM Treating Genetic Diseases Two main approaches to treating genetic diseases 0 Modifying disease phenotype Living with genotype Trying to figure out how to change environment to make that less severe o Replacing the defective gene You would no longer have disease if you could replace defective copy with good copy gene therapy 0 Modifying the disease phenotype can be done in 3 ways Restricting the substrate In As in PKU reducing phenylalanine in the diet Metabolic inhibitors 2 Such as drugs that can target specific proteins U Ex drugs to inhibit stuff Supplying the missing protein Blood factor VIII in hemophilia 2 Induce missing protein with drug then you can provide functioning protein by injection 0 Gene therapy Supply the missing aees by inserting a new gene that will be expressed in the patient The challenges Must find appropriate vector ensure precise insertion into host DNA ensure appropriate expression and select cells to target 2 vector agent that carries good gene into human cell integrate into human genome viruses do this naturally but our cells reject viruses 2 stuff needs to be expressed at the appropriate place 2 if you need the gene in a certain place how do you get it there Ex vivo gene therapy In In cell culture 2 Take cells that are effected add the gene that you want back to them then you are growing cells that HAVE the good gene you can check those cells and see if any become cancerous like you don39t want to inject the ones that are gonna be cancerous 2 Then you can put the cells back into the main thing Gene therapy problems 2 Viral infection can be dangerous in sick patients Jess Gelsinger died due to a gene therapy trial using adenovirus 2 SCID Severe Combined Immunodeficiency 10 SCID patients successful treated using viral vector 4 patients developed leukemia due to the particular gene that was used additional successful trials have avoided oncogenic genes CHAPTER 16 Regulation of Gene Expression Virus life Bacteriaphages and HIV retrovirus Regulation of Gene Expression in Prokaryotes Lac operon Catabolite repression Trp operon Gene Regulation in Eukaryotes Promoters enhancers Regulator and activator proteins Chromosome inactivation Epigenetic gene regulation Small regulatory RNA molecules Viruses and Human Health Spanish flu 1918 1919 deady plague killing 50 mil 0 Effected young people too HIV virus 0 New HIV infections have been dropping 0 Global death from AIDS are also dropping 0 People living with HIV is increasing because they are not dying Virus 0 Mostly consist of nucleic acid DNA or RNA and capsid coat protein Viruses always need a living host to reproduce Often they kill host cell after viral replication Viruses have no metabolism Some animal viruses have membrane coat Because they use host cellular machinery it is difficult to block replication with drugs like antibiotics 0 Best defense is vaccine The lytic cycle a strategy for viral reproduction o Infect one cell go in make a bunch of copies like 500 then all of those copies can go pop out and infect more stuff 0 Lysogenic cycle Can also immigrate into genome then they hideout allowing themselves to be replicated Then they can switch back and forth between lytic and ysogenic making tons of copies and stuff of itself 0 Retrovirus o GenomeRNA o It attaches to certain cells 0 HIV virus attaches to T ces part of immune system Attacks immune system 0 It does this by interacting with specific things on T cells specific interaction 0 Reverse transcriptaseenzyme RNA9DNA o Vira DNA then VIRAL DNA can integrate into HOST DNA infects T helper ces uses its receptors CD4 causing another receptor to grab hold of the envelope OOOOO
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