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46 Cards in this Set
- Front
- Back
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What are the required properties of genetic materials?
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1.reliable storage of all development
2.controlled expression of stored information 3.accurate replication 4.capable of some change |
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What is the transforming principle?
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Griffith isolates 2 strains of Streptococcus pneumoniae.
1.s strain - virulent - isolate live s out of blood 2.r strain - avirulent - 0 isolated 3.heat- killed s strain - 0 live bacteria 4.heat killed s strain and r strain - isolated live s strain Conclusion: there must be a transforming principle Avery & Colleagues Used lysate from s and systematically subtracted components using enzymes 1.lysate - protein = transformation 2.lysate - protein - RNA = transformation 3. lysate - protein - RNA - DNA = 0 DNA is transforming principle Genes are made of DNA |
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Hershey-Chase Experiment
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Phage studies - knew phage infect bacteria to make more phage - made of 50% protein and 50% DNA
used radioactivity to label either phage DNA or protein infect bacteria with phage and tracked where radioactivity went only DNA transferred into host must be directing synthesis of new phage |
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Nucleotide Chemistry
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nucleotides are the building blocks of nucleic acid
3 parts: pentose sugar nitrogenous base phosphate connected via phosphodiester bond |
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Purine vs. Pyrimidine
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purine - nine-membered double-ring
pyrimidine - six-membered single-ring oligonucleotides - 20 or less nucleotides in a chain polynucleotides - more than 20 |
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Chargoff base composition
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used sensitive techniques to quantify amount of each base in DNA found from different species
A=T G=C Purine (A + G) = Pyrimidine (T + C) |
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X-Ray diffraction
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Wilken & Franklin
photo 51 |
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6 characteristics of Watson/Crick model
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1.right-handed helix
2.anti-parallel strands 3.sugar phosphate backbone on outside; bases extend inward (more hydrophobic) 4.complete turn every 10 bases (10.4) 5.alternating major and minor grooves 6.arranged in complementary pairs bonded via h-bonds |
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Analytical techniques for DNA
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a) determining concentration & purity
1.uses absorbances of uv light 2.measure with a spectrophotometer b) determining melting temperature 1.Tm = 50% of the strands have separated ("denatured") 2.monitored using UV absorbance 3.informative about DNA length and sequence c) Finding sequence of interest 1.use molecular hybridization - one strand from one source and one strand from another |
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Possible models of DNA replication
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conservative - a complete new daughter helix
semiconservative - you peel apart the strand and replicate a daughter strand each dispersive - separate by segment and distribute evenly between the new daughter |
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Meselen-Stahl Experiment
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studied replicating e. coli
used different isotopes of N (14 or 15) to track parent and daughter DNA strands conclusion = semiconservative model |
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DNA polymerase
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enzyme responsible fro majority of process
3 types I = DNA repair and some replication II = DNA repair III = majority of DNA replication common properties of all types 1) can only add to an existing strand, can't start one 2) can only polymerize in a 5' to 3' direction - proof reading activity - if it adds the wrong nucleotide it can fix its mistakes, also each nucleotide must carry its own energy - cannot occur in reverse 3) has 3' to 5' exonuclease activity - cuts the phosphodiester bond and removes nucleutides |
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How replication occurs: prokaryotes
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FOR EACH STRAND
1) separate strands (helicases) 2) start new strand by making RNA primer (pimase) 3) add to primer, polymerizing DNA (DNA pol III) 4) remove RNA primer and replace w/ DNA (DNA pol I) 5) make final phosphodiester bond (ligase) ON CHROMOSOME occurs bidirectionally replicating both strands at the same time Initiation phase strands open at origin replication bubble forms replisome (DNA pol III and additional enzymes and proteins) assembled at each fork Elongation phase replisomes move in opposite direction Each strand at each fork replicated differently because a dingle DNA pol III at each fork, lagging strand must loop or fold over to allow replication leading strand = continuous lagging strand = discontinuous |
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Replication in Eukaryotes
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3 main differences
1) many more polymerases 2) multiple origins per chromosome (25000 in human genome) 3) has to replicate chromosome ends (linear) ends called telomeres cannot complete replication on the lagging end; every time cells divide, telomeres shorten - when last primer is removed we can never fill it in telomerase = enzyme used in embryonic cells to maintain chromosome length: part protein part RNA, adds nts to the end of the chromosome, uses RNA as a template |
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Prokaryotic chromosome?
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found in a compact region called the nucleoid where it is maintained in a supercoiled state (gyrase)
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Eukaryotic chromosome?
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most familiar with metaphase chromosome; two sister chromatids connected at a centromere
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Haploid vs. Diploid?
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Haploid - 1 of each chromosome, in humans this is only in the gametes
Diploid - 2 of each (1 from each parent) called somatic cells; each pair is "homologous" |
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karyotypes?
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the arranged display of chromosomes in a a mitotic cell, each identified by size, placement if centromere, and banding pattern (G dye)
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Two types of chromatin?
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Chromatin - DNA and all associated protein
Euchromatin - majority of chromatin, contains most of genes, highly organized Heterochromatin - variable in structure (more condensed), contains few genes, very hard to sequence, specialized parts of chromosome and genes that are silent (no gene activity) |
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4 levels of chromatin organization?
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1) nucleosomes - DNA helix that is wrapped around the histone (protein)
2) solenoid - (30 nm chromatin fiber) coil nucleosomes into solenoid, histones are close enough to start associating and help coil 3) looped domains (300 nm chromatin fiber) 4) metaphase chromosome can be remodeled to affect gene activity - more condensed = genes "off" and less condensed = genes "on" |
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Spontaneous vs. Adaptive hypothesis of mutation?
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Spontaneous - 1st random mutation, then pressure
Adaptive - 1st pressure, then targeted mutation Luria and Delbruck's fluctuation experiment - spontaneous complexities: mutation hot spots (e.g. repetitive DNA, tandem repeats, methylated CG sequences), SOS response in bacteria (error-prone enzyme used), somatic hypermutation in antibody (secreting B cells) |
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Types of mutation?
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Chromosomal location: x-linked vs. autosomal
Cell type: germline vs. somatic Effect on protein function: loss-of-function (recessive) vs. gain-of-function (dominant) Characteristic changed: morphological vs. nutritional/ biochemical vs. behavioral Molecular change in DNA: substitution vs. deletion Effect in translation: silent, neutral, missense, nonsense, and frameshift |
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Effect in translation: silent
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one aa to same aa
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Effect in translation: neutral
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one aa changed to equivalent aa
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Effect in translation: missense
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one aa changed to non-equivalent aa
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Effect in translation: nonsense
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one aa changed to STOP
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Effect in translation: Frameshift
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Insertion or deletion that changes the frame
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Mutation rates?
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surprisingly low for all organisms, varies between species: so much DNA, such a big genome, the chance of a mutation making a serious impact is so low; varies between genes in same organism
KEEP IN MIND: rates are based on phenotypic change, proteins vary in sensitivity to change, protein under different selective pressure |
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Causes of mutation?
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Spontaneous mutation:
DNA polymerase errors - wrong nt attached and not fixed, replication slippage (slip forward deletes repeats in new strand, slip backward adds more repeats in new strand), tautomeric shift (tautomer - alternate form of base), spontaneous damage to nts (depurtination - break covalent bond between sugar and purines, deamination - losing an amine group) Induced mutation: Radiation - UV (causes pyrimidine dimers; bulky lesions) and Ionizing radiation (causes chromosomal ds breaks), chemical mutagens |
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DNA repairs types??
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1) DNA pol. proofreading - fixes "mismatches" immediately
2) photoreactivation - fixes pyrimidine dimers, mediated by PRE, absorbs blue light & breaks bonds 3) excision repairs - "cut" out damage on strand & fill with new DNA: BER (fixes damaged base), NEW (fixes dimers & bulky lesions) 4) mismatch repairs - fixes mismatches missed by DNA pol. 5) ds break repair - fixes chromosomal breaks, detects blunt ends: end joining of homologous repair (missing nts copied from homologous chromosome and ends connected) |
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Transposable Element?
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a section of DNA that can move
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TEs in prokaryotes?
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Insertion sequence
transposase = enzyme that mediates transposition inverted repeats = sequences recognized my enzyme Transposons bigger and contain several genes, probably 2 ISs trapping DNA between them, can move genes onto plasmid |
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TEs in Eukaryotes?
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mcklintock - studied maiz and correlated chromosome break with color change
Activator (Ac) - mutated IR, functional transposase Disassociation (DS) - functional IRs, mutated transposase RS jump in and out - function w (cell purple) and non-functional w (cell white). kernel mottled when dissasociator jumps in and out |
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3 steps of transposition?
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1) RNA copy made by transcription
2) DNA copy made by a reverse transcriptase 3) DNA copy inserted in new location |
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Constitute vs. Inducible expression?
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Constitute: all the time, in pro. determined by how well sigma factor binds promoter
Inducible: not all the time, turned on or off by stimulus, usually involves trans-acting factors whose affinity for DNA changes, under positive or negative regulation |
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+ regulation vs. - regulation
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+ activator promotes transcription
- inhibitor discourages transcription |
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Recombinant DNA - restriction enzymes?
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cut DNA helix at specific sequence (restriction site), make blunt or staggered cut (staggered - ss overhangs), DNA cut w/ same restriction enzyme easily recombined (ligase seals gap on each strand)
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Cloning?
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making many copies of one sequence
requires: host cell - will replicate DNA vector - will carry DNA into host and allow recognition |
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Cloning vectors?
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vectors differ depending on host cell
must have: one or more unique restriction site and origin of replication selectable marker genes |
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Blue/White screening?
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put in bacteria and plate on media containing color changing B-gal substrate - blue determines if bacteria is making B-gal
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DNA sequencing?
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identifies nts and their order in a DNA sample
mix together: 1) DNA to sequence 2) primer 3) DNA pol. 4) all 4 dNTPs (high concentration) 5) all 4 ddNTPs (low concentration) *DNA replicated many times a) if ddNTP incorporated - rxn stops b) DNA frag. of different lengths c) each frag. ends w/ diff. color indicating identity of last nt d) machine sorts frag. by size and detects color |
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Genomics?
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studying entire genome of an organism
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Proteomics?
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studying genome-wide patterns of gene expression
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Bioinformatics?
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developing tools for analyzing large quantities of biological data
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Sequencing genomes?
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shotgun sequencing:
sheer DNA into random but overlapping fragments, sequence each fragment, finds where contig overlaps and assemble into continuous sequence 2 methods: 1) HGP's - ordered-clone shotgun sequencing: make physical map of each chromosome, sheer DNA & clone into plasmids, place clones in order based on "landmarks," shotgun sequence each clone 2) Venter's - whole-genome shotgun sequencing: sheer DNA & clone into plasmid, shotgun sequence each clone, use powerful algorithms to assemble all contigs for entire genome |
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Genome annotation?
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assigning function to sequence
- perform a BLAST search: enter query sequence, tools compare it to all sequences in database, hope you find a match - try to identify open reading frames (protein coding regions), check all 3 frames on each strand, correct one has a start & stop in frame easier in pro. because of introns - interrupt protein coding regions - an because of size of genome |
