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31 Cards in this Set
- Front
- Back
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Who are some discoverers of the chemical nature of genetic material?
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1869 Miescher's discovery of DNA led to conflicting proposals; discovered nuclein from WBC and fish sperm
1880 Zacharias said extracting DNA from cells causes chromosomes to disappear Avery: -DNA = bacteria -first to discover DNA carries genetic information -S strain capable of transformation -Determine whether protein/RNA/DNA is transferred -use RNase, DNase, protease to eliminate transformation ability Hershey and Chase: -DNA = viruses -virus has coat and uses prong to inject into bacterial cell wall, protein coat leaves (marked with radioactive sulfur so you can see leaving), DNA now in cell and replicates as a virus Chargaff: -A = T, G = C -higher temp = more G/C, stronger bonds |
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What is DNA structure? How is it packages? What are properties of its nucleus?
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Structure:
-DNA organized in genomes -Watson Crick discovered double helix -restriction endonucleases cleave DNA at specific sites Packaging: -bacteria package DNA in chromosomes and plasmids -nucleosomes are the basic unit of chromatin structure -histone octamer forms the nucleosome core -eukaryotes package some DNA in mitochondria and chloroplasts Nucleus: -double membrane envelope -matrix and lamina support structure -nucleolus involved in ribosome formation |
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What is the Griffith Experiment?
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identified genetic transformation in pneumococcus
S (smooth) strain has capsule - evades immune system --> kills R (rough) strain NO capsule - immune system attacks --> OK Heat killed S --> OK Heat killed S + living R --> kills (find S living in blood, transfer of DNA from killed to living (TRANSFORMATION)) |
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What is the Watson and Crick DNA model?
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1953
double helix: -right handed -normal = BDNA -dehydrated = ADNA (wider) -ZDNA = places of transcription, relieves torsional strain -major groove -minor groove -antiparallel orientation of two DNA strands -complementary strands -pyrimidine-purine pairing (A with T, C with G) -5' to 3' orientation |
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How are DNA interconverted between relaxed and supercoiled forms?
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Topisomerase I:
-catalyzes stepwise relaxation of supercoiled DNA by cutting DNA DNA Gyrase: -Type II -can induce as well as relax supercoiling -requires ATP to generate supercoiling but not to relax |
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How can DNA double helix be separated by denaturation and rejoined by renaturation?
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DNA melting temperature (Tm):
-one half of 260 nm absorbance change is achieved -higher for G/C because triple bond PCR: -DNA amplification, copies DNA without bacteria (Kary Mullis) Northern Blot: -RNA Souther Blot: -DNA Renaturation doesn't work like a zipper, goes back to same structure negative charges on phosphates repel, allow breathing of double helix |
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How is DNA detected by nucleic acid hybridization?
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hybridization with a radiolabeled DNA probe
denatured by heating to 95 C yielding single-stranded molecules probe is added and temp goes to 65 allowing complementary DNA strands to renature by pairing with each other, this can be detected as radioactive double stranded molecules |
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How are stringent vs. reduced stringency hybridization conditions different?
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stringent (42 C):
-single stranded DNA for one gene -only that gene forms stable helix reduced stringency (35 C): -mixture of many genes -more than one form stable helix at 35 C it binds slightly to what matches it, more ambiguous, shows that small temp change makes big difference not used anymore |
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What is Membrane-hybridization assay?
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detects nucleic acids in RNA and DNA, radiolabeled probe can be either DNA or RNA
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What do restriction enzymes do?
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isolated from bacteria that can cut DNA usually at a unique palindromic end, go into major groove
important tool for cloning and gene mapping sticky ends = 3' overhangs blunt ends = 4 base pair cutter allows for recombination --> cut different DNA with same restriction enzyme and use ligase and low temp to bring it together *ECO R1 is an example that cuts where ever 5' GAA TTC 3' is present |
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What is Restriction Enzyme Mapping?
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used to order in which a set of restriction enzyme fragments is arranged in a molecule
steps: -digest DNA with one enzyme, separate by gel electrophores -from gel, isolate bands of DN, and cut DNA with another restriction enzyme -separate double-digested DN, out by gel electrophoresis again |
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What is the process of gel electropheresis of Nucleic Acids?
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-loaded into a gel (agarose or polyacrylamide) and subjected to electric potential across gel will migrate to anode
-nucleic acids are inherently negatively charged -separate based on size, smaller move faster and further -have to add a dye on DNA to see it on gel, example is ethidium bromide |
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What is the significance of Dideoxyribonucleotides?
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A DNA strand terminating in dideoxynucleotide cannot be elongated because a 3' OH is necessary for polymerization
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What did Frederick Sanger win Nobel prizes for?
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Frederick Sanger had 2 nobel prizes; one for gene sequencing:
-between Sanger, Gilbert, and Berg -added a dye-labeled dideoxynucleotide with DNA polymerase, primer, and deoxynucleotide -a color came up each time DNA sequencing prematurely terminated because of the dye -fragments then separated by gel electrophoresis -camera detects color fragments as they pass through gel, plotting the sequence of bases -smallest fragments come off first from 5' end 2nd was for structure of insulin |
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What are proteomes and a technique used to distinguish peptides?
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proteomes:
-structure and properties of every protein produced by the genome technique: Mass Spec - high speed, sensitive technique separates peptides based on differences in mass and charge |
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What are Single Nucleotide Polymorphisms?
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SNPs (Snips)
-base sequence variation between individuals that result from point mutations -only 0.3 % differences in bases from person to person -might be useful for predicting disease or phenotype in people |
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What is a unique difference about renaturing between bacterial and mammalian DNA?
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Mammalian DNA renatures much faster
ex. Cow DNA much faster than E Coli DNA 2 classes of mammalian DNA observed: 1. Fast annealing - multiple copies of repeated sequences 2. Slow annealing - fraction encodes for nonrepeated DNA Newt Amphibian has a huge genome, lets of repetitive DNA |
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What are the categories of repeated sequences in Eukaryotic DNA?
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1. Tandemly repeated DNA:
-mutiple copies arranged next to each other in a row -10 - 15 % mammalian genome -centromeres and telomeres are tandem repeats ex. satellite DNA, minisatellte, microsatellite 2. Interspersed repeated DNA: -scattered around genome -25-40 % mammalian genome -Alu family is large portion -transposable elements (self-replicating parasites) |
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What are two families involved in Scattered Repetitive DNA?
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Alu family in higher primates:
-10 % of human genome -300 bps length -Alul restriction site present in each member of Alu (Arrthrobacter luteus) family L1 family: -long repeates -100000 copies per genome -transposons or 'jumping genes' replicate themselves in genome, leaving copies behind -both have been implicated as cause of hereditary diseases by retrotransposition (DNA to RNA, back to DNA) -abberant recombination events of dispersed repeats also causes genetic diseases (colon and breast cancer; familial hypercholesterolemia) |
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What are the Microsatellite DNA diseases?
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Huntington's disease:
-contains excessive amount of tandemly repeated CAG (glutamine) sequences Fragile X syndrome: -major cause of mental retardation in males - CGG (arginine) repeats Myotonic dystrophy: -muscular disease - CTG (Leu) repeats |
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What are the different levels of chromatin packing?
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DNA double helix - 2 nm
Nucleosomes ("beads on a string") - 10 nm 30 nm chromosome fiber called solenoid, requires H1 and can be folded into loop domains Looped domains - 300 nm, attached to chromosomal scaffold Heterochromatin - 700 nm, chromatin fibers contain 6 nucleosomes per turn highly condensed, duplicated chromosome of dividing cell - 1400 nm |
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What is the structure of a Chromatosome?
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contains two full turns of DNA (166 base pairs) locked in place by one molecule of H1
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What is the organization of chromatin in nucleosomes?
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DNA is wrapped around histones in nucleosome core particles and sealed by Histone H1
Nonhistone proteins bind to linker DNA between nucleosome core particles |
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What is Heterochromatin and Euchromatin?
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Heterochromatin:
-highly folded, usually transcriptionally inactive sometimes Euchromatin: -loosely packed DNA -has most transcriptionally active genes |
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What are characteristics of the DNA packaged in mitochondria and chloroplasts?
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-devoid of histones and are circular
-most polypeptides are components of multimeric proteins that contain subunits encoded by nuclear genome |
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What is the Nuclear Pore Complex?
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NPC
inner and outer nuclear membrane fuse together forming a channel lined with a large protein complex ring structure with 8 subunits in octagonal pattern has transporter granule that moves proteins in and out cells have about 3000-4000 pores passive diffusion of small molecules (NO proteins) active transport of large proteins and RNA: -importin takes proteins in -exportin takes proteins out |
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What are properties of the nuclear matrix and nuclear lamina?
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matrix:
-insoluble fibrous network after chromatin has been removed -skeleton for chromatin fibers lamina: -thin, dense meshwork line inner surface of inner nuclear membrane -made of intermediate filaments made of proteins called lamins |
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What are constitutive and facultative heterochromatin?
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constitutive:
-highly condensed at all times -centromeres and telomeres are two major regions that form constitutive facultative: -specifically inactivated in specific cell types -low in embryonic cells and higher in differentiated cells |
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What does the nucleolus do with ribosomes?
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membrane free organelles in nucleus
DNA sequences encoding rRNA reside in nucleolus -chromosome 13, 14, 15, 21, 22 contain nucleolus organizer region (NOR) - stretch of DNA carrying multiple copies of rRNA genes disappears after mitosis NORs re-establish nucleoli at end of mitosis |
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What is DNA fingerprinting?
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Restriction fragment length polymorphisms (RFLPs) can be used to identify individuals - change in palindrome
Variable number tandem repeats (VNTRs) can also be used (criminal cases) PCR can amplify DNA from small amounts of samples |
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What is RT-PCR?
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Reverse transcriptase-PCR
start with mRNA convert to cDNA then make dsDNA then do regular PCR 3' end of mRNA stablizes it and prevents degradation, capped at 5' end |
