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84 Cards in this Set
- Front
- Back
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differences between DNA and RNA
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DNA: deoxy, ds, thymine
RNA: ribose, ss, uracil |
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ds, helical, and antiparallel chain structure of DNA
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ds: Watson and Crick
helical: helicase and topoisomerases antiparallel chain: leading and lagging strands |
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recombinant DNA
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1. pieces of DNA from different origins ligated together to form a single piece
2. uses restriction enzymes, DNA ligase, and vectors 3. creates a piece of DNA that can be autonomously replicated in a host cell (bacteria or eukaryote) 4. isolate large quantities of DNA of a particular sequence 5. create libraries, or collections of DNA pieces that represent an entire genome (genomic library) or the entire complement of expressed genes (cDNA library) |
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restriction endonucleases
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cut dsDNA at specific palendromic sequences
in cutting they leave either a 5' or 3' overlapping ends (called sticky if staggered and blunt if not) do not cut RNA in a DNA-RNA association |
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blunt end ligation
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if a restriction enzyme generates a blunt end the ends can be ligated together but must maintain 5' and 3' correctly
(don't need to be cut w/ the same enzyme) |
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how to check if restriction digest worked
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separate DNA by size using an agarose or acrylamide gel electrophoresis
since DNA is negatively charged it migrates toward the cathode smaller pieces travel faster adding ethidium bromide allows for visualization |
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cloning vectors
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essential for manipulating DNA
1. required to propogate and increase the amount of cloned DNA 2. place unknown DNA into a vector which replicates autonomously from the host cell and is easily retrieved for large-scale purification types of vectors: 1. bacteriophage (used for genomic libraries) 2. plasmids (for cDNA libraries and subcloning) 3. cosmids, bacs, and yacs, used for genomic libraries |
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essential properties of cloning vectors
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foreign DNA can be easily inserted and vectors containing foregin DNA can be selected and distingusihed from vectors which did not incorporate
they replicate autonomously in the host cell (this is key) DNA is easily separated from host cell DNA |
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common plasmids
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pBR322 and pUC
common features of vectors: 1. antibiotic resistance 2. unique endonuclease restriction sites for inserting foreign DNA 3. color selectionwith pUC which helps to distinguish vectors that have succesfully ligated foreign DNA (blue=no insert, clear/white=yes insert) |
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polylinker clning site
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used to facilitate cloning of foreign DNA
these enzyme sites are unique (present only 1 time in this location in the entire vector) pUC has these |
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steps of using a cloning vector
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1. restriction of DNA w/ endonuclease restriction enzymes
2. ligation of DNA fragments into plasmids 3. transformation of plasmids into bacterial cells 4. selection of bacteria that contain the DNA plasmid (using antibiotics or color change) |
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cosmids
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cloning vectors that can incorporate large amounts of foreign DNA (up to 45 kb)
antibiotic resistance can be used to select bacteria w/ cosmids cosmids enter the bacteria through infection from a bacteriophage (phage must have 38-52 kb of DNA to replicate) |
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problems with cosmids
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cosmid-cosmid ligation (concatenation of the vector)
insertion of multiple pieces of foreign DNA instability of foreign DNA differntial growth of clones |
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YACs, BACs, and PACs
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YAC=yeast artificial chromosome
BAC=bacterial artificial chromosome pACs=p1 phage artificial chromosome used to generate genomic libraries antibiotic resistance YAC: 100-500 kb BAC: ~150 pAC: ~150 |
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bacteriophage
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head w/ genetic material, tail and tail fiber which allows for injection
single bacterium is only infected by a single phage (and the opposite is true) plaques form on lawn of bacteria where lysis of cells has occurred |
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summary of cloning vectors
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plamids: <10kb, good for sucloning
-antibiotic resistance, color selection -cDNA phage: larger inserts (~20 kb) -genomic libraries cosmids, YACs, BACS, pACs -very large inserts (45-250 kb) -antibiotic resistance -genomic cloning |
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genomic library
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contains all the DNA w/in an organism including exons and introns, promotors, 5' and 3' flanking sequences, and intergenic sequences
use partially digested genomic DNA to make library so that overlapping DNA fragments can be isolated and linked together in a long contiguous sequence |
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genomic library construction
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partially enzyme-restricted DNA can be isolated from agarose gels, purified and ligated to vectors (bacteriophages, cosmids, YACs...) to create a genomic library
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cDNA libraries
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reflection of the mRNA that is expressed in a particular tissue a at a paritcular time
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method for making cDNA
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1. isolate polyA+mrna
2. anneal a primer to the mRNA 3. make the first cDNA strand using mRNA as a template, a primer which is bound to the mRNA, dNTPs, and a reverse transcriptase 4. synthesize the second cDNA strand using DNA polymerase (Klenow fragment) 5. RNAse H digestion of the RNA component 6. add "linkers" to the ends of the cDNA and ligate a vector |
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DNA denaturation and reassociation
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if DNA is heated its strands will separate (denature)
if cooled down, DNA strands will reassociate (hybridize) forms the basis for generating probes in Southern and Norther blots, screening libraries, and diagnostic testing ((two complementary bases will separate not the phosphodiester backbone) |
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COT curves
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cot=1/k
describes the renaturation of nucleic acid depending on random collision with complementary strands denaturation: increases with heat or high pH renaturation: increases w/ increased salt concentration (low pH) COT depends on: 1. amount of DNA in rxn (more DNA the greater the COT) 2. salt concentration (more salt=lower COT) 3. repetitiveness of DNA (more repetitiveness decreases COT) based on the fact that ssDNA absorbs light better than dsDNA |
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eukaryotic cot curves
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fast: 25% of genes that are hightly reptitive
intermediate: 30% of gene medium repetitiveness like ribosomal DNA slow: 45% of genome (genes b/c there is hardly any repetitiveness) |
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probes
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necessary for DNA-DNA and DNA-RNA hybridizations
2 primary sources: 1. closely related DNA 2. synthetic oligonucleotides "washing condtions" low salt and high temperature=high strigency |
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DNA-DNA hybridization
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1. DNA is denatured (usually w/ a basic solution)
2. fixed to nylon paper membrane 3. probed w/ radiolabeled or fluoreseccently labeled DNA ss probe 4. probe hybridizes with DNA that is fixed and has significant nucleotide homology (>80%) 5. binding can be detected through autoradiography or fluorescence |
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DNA-DNA hybridization to identify desired DNA fragments in a genomic or cDNA library
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1. bacteria containing the vectors w/ foreign DNA are plated on agar medium
2. nitron/nitocellulose fileter paper is put on top of them 3. paper treated with based to denature DNA 4. DNA is fixed to the paper 5. hybridization with a labeled probe 6. following autoradiography, the paper can be matched with the original bacterial colony and then the deisred bacterial clone can be isolated and grown for a large scale DNA prep |
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SDS-page
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PAGE=polyacrylamide gels that are used to separate proteins via electrophoresis
SDS is often added to confer a larger negative charge to proteisn so that they are truly separated by size ane not charge molecular weight markers (in kD) are indicated on the side bsaed on how far the protein traveled |
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isoelectric focusing
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separates proteins based on their charge (isolelectric point)
based on pH |
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2-D electrophoresis
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combines SDS-PAGE and isolectric focusing to get separation based on both
1st charge than MW |
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Mass Spec
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after running a 2-D gel, you could take a protein from a specific location and digest it
then use a mass spec to fingerprint for the peptide composition |
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expression of cloned genes
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fusion proteins: proteins coupled with other chemicals to facilitate isolation and purfication and identification of desired proteins
-often expressed in bacteria transgenic animals: planned genetic modification on genome for varoius purposes such as 1. animal modles of human disease 2. testing of gene therapy 3. product development vector of choice is: replicant-deficient adenovirus b/c it 1. infects dividng and nodividing cells 2. remians epichromosomal 3. is stable limitations: 1. method of delivery 2. persistence of gene expresison 3. regulation of gene expression 4. widespread transfer of vector w/in the host |
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RNA interference
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used to silent a gene
1. siRNA duplexes are transfected and then they become incorporated into the RISC which leads to degradation of target gene expression 2. transfected plasmid vectors reach the nucleus for transcription of shRNA this is produced and then trnasmpoted to the cytoplasm where it is incorporated into RISC 3. viral vectors encoding siRNA or shRNA bind to target cell via a rectpor and followed by receptor-mediated endocytosis |
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natural gene interference
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siRNA (small inhibitory RNA)
shRNA (small hairpin RNA) miRNA (micro RNA) end game is the same: decreased expression of a desired RNA or protein by degrading endogenous mRNAs or blocking translation -as a therapeutic agent, these RNAs are being used in the treatment of many diseases -cholesterol increases the uptake of siRNA into different cells -uptake can also be targeted by binding something to the siRNA that will bind to a specific recptor |
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southern blots and DMD
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Duchenne's Musuclar Dystrophy
DNA from different pts is electophoresed on a gel, transferred to nitrocellulose, and then examined with probes the probe will hybridize to normal DNA but not to DMD pt's if the sequence is missing |
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cDNA and genomic DNA
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all of cDNA sequence should map to genomic DNA fragments but only some of genomic DNA will hybridize to cDNA sequences
2. exons are mapped from genomic DNA by taking the genomic fragments and hybridizing them to Nothern blots (if a transcript is detected then the genomic DNA has an exon) |
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ply A+ RNA
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used to increase the sensitivity of detecting mRNAs on nothern blots
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DNA sequencing
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often used to deduce the function of a gnee/piece of DNA
most widely used method of DNA sequence-Sange rmethod-uses dideoxy chain terminators |
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process of DNA sequencing
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1. clone foreign DNA in a vector
2. anneal a primer 5' of the cloned foreign DNA 3. set up 4 tubes with ddNTPs, dNTPS, DNA polymerase (Klenow fragment), and vector/foreign DNA 4. allow rxn to occur for 40 min. at 37 degrees 5. run acrylamide gel and read DNA sequence in 5' to 3' direction |
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DNA sequencing output
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nucleotide seequnce can be read from the bottom up in the 5' to 3' direction
this process has been automated using fluorescence to figure out w/ nucleotides are in what order but you still have to figure out the reading from yourself (usually the longest reading frame is the right one) |
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DNA sequencing and cystic fibrosis old school
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this used to be done to determine whether or not there were partiuclar mutations in cystic fibrosis patients
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DNA sequencing and cystic fibrosis now
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use an allele-specific oligonucleotide to test for mutant gene (also use a normal oligonucleotide gene probe)
readout is called an autoradiogram |
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DNA microarrays
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purpose:
1. to examine the expression of thousands of genes in a single asssay 2. to QUANITATIVELY relative gene expression profile betwen two populations of cells or tissues |
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method of DNA microarrays
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spot 1000's of DNA gene identifying sequnces onto a matrix (i.e. glass clide)
competitively hybridize fluorescently labeled cDNAs from different cell populations, assay the fluorescnet readout and analyze w/ a computer |
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typical DNA microarray sequence
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1. prepare DNA chip using your target DNA of interest
2. isolate and purify mRNA from your different sources 3. reverse transcribe the 2 mRNA populations into 2 distinct cDNA using two different fluorophores (red and green, e.g.) 4. generate a hybridization solution containing equivalent amounts of both fluorescently labeled cDNA 5. incubuate and hybridize mix with the DNA chip 6. scan the bound cDNA using laser technology 7. store data in computer and analyze |
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microarray analysis
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each spot on an array is associated w/ a particular gene
intensity of color gives an estimate of the amount of expression |
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PCR
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polymerase chain rxn
used to amplify specific DNA fragments millions of times for research of diagnositc purposes can also be used to amplify RNA w/ modifications to technique |
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essential requirements for PCR
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1. synthetic oligonucleotide primer (about 20 nucleotides each) that are complementary to regions on opposite strands and are oriented with their 3' hydroxyls toward each other
2. target sequence in the DNA (or RNA) that lies btwn the pairs of primers which can be from 100-5000 nucleotides in length 3. thermostable DNA polymerase (usually Taq polymerase) that can withstand 95 C or higher 4. the four deoxyribonucleotides |
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steps of PCR rxns
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1. denaturation by RAISING TEMPERATURE (~1 min)
2. renaturation by cooling temperature slowing (~2-3 minutes) to 55C which allows primers to base pair with their complemntary sequences in DNA 3. synthesis: temperature is raised to 75 or so so that Taq DNA polymerase can function -DNA synthesis is initiated at the 3' hydroxyl end of each primer (~2 minutes) |
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one use of PCR
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can be used to determined if deletions of exons or alternative splicing occurs w a primary trnascript (reverse transcriptase is needed of starting with RNA)
by using 2 primers corresponding to nonadjacent exons and amplifying the RNA, one can examine the products to detremine if exons are missing or multiple mRNAs are produced |
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diagnostic PCR
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perform PCR and electrophorese on the products on an agarose gel and compare to normal
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insulin: T to R conversion
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T to R transition involves a conformational change in the first 9 residues of the beta chain
-three distinct conformational states: T6, T3R3, and R6 -go from extended conformation in Tstate to alphahelical in Rstate |
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frayed conformation of insulin
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T3R3
creates a hydrophobic pocket (phenolic pocket) in the Rstate alterations to the first 9 AAs of the beta chian affect this hydrophobic ligand binding cavity frayed state: end of the alpha helix is not helical |
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R form of insulin
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can bind hydrophobic molecules like methylparabin
low pH favors R form (opposite of Hb) each subunit can bind a 6 hydrophobic ligand |
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tunnel leading to anion binding site in R state of insulin
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helix side chains make up the tunnel (Phe, Asn, Leu)
zinc coordinates to 3 his residues at the bottom cavity primarily hydrophobic |
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methylparabin binding to insulin
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binds to the dimer-dimer interfaces of the T6 hexamer
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gleevec demonstrates
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rational drug design
protein kinase inhibitor therapy for CML |
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philadelphia chromosome
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shortened chromosome 22 resulting from a translocation between chromosome 9 and 22 produces BCL-ABL oncogene
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BCL-ABL
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hallmark of chronic myeloid leukemia (CML) ans a subset of acute lymphocytic leukemia
abnormal protein molecule causes bone marrow to release large numbers of WBCs |
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role of ABL
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constitutively activates tyrosine kinase
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mechanism of gleevec
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first member of a new class of targeted therapy
specifically inhibits tyrosine kinase developed by rational drug design many ppl develop resistance due to mutations in ABL when kinase autophosphorylates itself it blcoks NERs into the nucleus--Gleevec blocks this autophosphorylation so it goes into the nucleus and induces cell death |
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heat of vaporization
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high due to the energy required to break H-bonds
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high dielectric constant
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water is polar and conducts electrical current
hydrophobic interxns key of protein folding |
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HIV
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infects and kills T cells
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anti-retroviral dideoxynucleosides
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these molecules carry no 2' or 3' hydroxyl so if incorporated into a growing DNA chain (2' deoxy) they wil terminate it
AZT and 3TC are examples |
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mast cells
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granules contain bioactive substances (histamine, proteoglycans--give metachromasia)
slow release of granule contents and cytokins regulates innate immune response rapid release of contents during an allergic rxn causes anaphylactic shock |
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scleroderma
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collagen fibers are denser and extend more deeply throughout the subcutaneous region
antifibrotic drugs are ineffective treatment is mostly supportive etiology is believed to involve changes in vasculature (hence Reynaud's Syndrome) |
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marfan syndrome zerba
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not only the strucutral defect of fibrillin-1 but also the fact that fibrillin-1 nomrally sequesters grwoth factor TGF-beta
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integrins and neutrophils leaving blood vessels
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at first they're in the inactive state but when they're activated they do allow the cell to leave
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leukocyte adhesion deficiency (LAD)
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neutrophils can't extravasate b/c they lack integrin Beta2
this is crucial defense against disease adhesion (tethering, rolling, and attachment) is crucial for neutrophil extrvasation |
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operator
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binds the repressor
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inducer
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de-depression b/c it binds to the repressor so that it can no longer repress
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requirements for transcription of lac operon
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absence of a repressor (via the inducer allactose) and
presence of an activator cAMP-CRP |
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is there a corelation btwn rates of trnascription and concentration of mRNA in euk cells
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NO
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isoforms
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alternative processing by alternative splicing or alternative polyadenylation sites
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mRNA half life
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can be impacted by the 3' untranslated sequence due to its effect on stability
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miRNAs
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transcribed as primary transcripts or w/in introns of other genes
sort of fit=blocking of translation perfect match-=destruction |
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mediator
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aka co=activator
non-DNA binding transcription factor complex that coordiantes the other Tfs |
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transcriptional initiation complexes
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typically involve multiple protein-protein and protein-DNA interxns
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hormones in trancriptional intiation complexes
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are a specific type of enhancer
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LCR
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highly complex regulatory element w/ multiple transcription factor binding sites
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therapeutic approahces for beta thalassemia based on epigenetic considerations
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inhibition of methylation of DNA at CG dinucleotides
inhibition of deacetylation of histones goal is to reactivate gamma globin genes in adult erythroid cells |
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fascin and fimbrin
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actin bundling proteins that cross-link actin
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intermediate filaments
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play a supporting or structural role
keratin and lamins |
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action of anti-malarial drug chloroquine
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increases the pH of lysosomes
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