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Microbial Genetics > History of DNA > Flashcards

History of DNA Flashcards

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1
Q

Mendel

A

1865- Plant Genetics paper

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2
Q

Friedrich Miescher (Swiss)

A

1869- purified nuclei of pus cells (later used salmon sperm)
gelantinous material - organic PO4 –> nuclein
70% protein (histones)
Discovery of nucleic acid

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3
Q

Albrecht Kossel

A

nuclein had organic bases, sugar

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4
Q

Richard Altman (student of Miescher)

A

1889 deproteinized yeast DNA –> started name nucleic acid

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5
Q

A. Ascoli

A

1901 uracil in yeast RNA

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6
Q

P.A. Levene

A

1908-1929 P.A. Levene - 2’-deoxy-D-ribose as DNA sugar
tetranucleotide theory
(very influential biochemist)

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7
Q

1910

A

DNA, RNA separate molecules

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8
Q

Levene & Bass

A

1931 - tetranucleotide theory
- 4 bases in a plane
- incapable of carrying genetic info
nucleic acids attracted very little attention

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9
Q

Fred Griffith 1928

A

Used Streptococcus pneumonia
transforming principle, rough and smooth (capsules) cultures
rough cells plus heat-killed smooth cells –> dead mouse
Initial reasoning: R cells restored viability to the S cells - not true
since he used cell-free extract of S cells and still changed R cells to S cells

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10
Q

Avery, MacLeod, McCarty (15 yrs later)

A

Partially purified the transforming principle
Demonstrated it was DNA, used modified known techniques
1. purified S culture DNA plus R cells –> plated it out
Results: culture with both S and R colonies isolated colonies,
replated - pure colonies
2. Attempted to use polysaccharide material as transforming material - no results

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11
Q

DNA extraction method

Avery, MacLeod, McCarty

A
  • very impure (other evidence required)
    1. chemical analysis, COP, nucleic acid
    2. physical measurements very viscous - nucleic acid
    3. Enzyme treatment
    not lost with proteolytic enzymes - trypsin chymotrypsin
    not lost with ribonuclease
    lost when treated with DNase
    no pure DNase - therefore, elaborate tests to confirm
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12
Q

Difficult time convincing the scientific community (published in 1944)

Avery, MacLeod, McCarty

A

Reasons

  1. tetranucleotide
  2. not enough variation with only 4 bases
  3. genes- chromosomal protein
    a. TP (transforming principle) was actually a contaminating protein
    b. DNA had a regulatory effect on capsule manufacture
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13
Q

Erwin Chargaff - 1952

A

Disproved tetranucleotide theory
Reason for tetranucleoside eukaroyotic cells - ATGC equimolar
used paper chromatography and UV absorbance
isolated DNA from wide variety prokaryotes
showed that molar concentrations of bases varied widely between different organisms
showed enough variation for DNA to be genetic material

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14
Q

Hershey and Chase

A

Blender experiment

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15
Q

Blender experiment

A

E. coli phage T2 (DNA in a protein shell)
DNA - phosphorus
Proteins - (methionine, cysteine) sulfur
grow with 32PO43- phosphate, radioactive “hot” DNA
35SO43- sulfate, radioactive “hot” proteins
hot or labeled phage 32P not 35S injected
blendor - tear off phage from bacteria see what goes inside

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16
Q

Blender experiment Proceedure

A
  1. label phage
  2. attach phage and centrifuge
  3. resuspend
  4. blendor then centrifuge
  5. 80% 35S in supernatant
    20% 35S in pellet (tail fragments attached)
    or
    70% 32P in pellet
    30% 32P in supernatant (breakage of bacteria during blending)
    or defective phage

Transfer Experiment (confirmation experiment)
one step further - isolate phage progeny
half 32P was transferred to progeny, no 35S

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17
Q

Structure of the DNA molecule

A

Early experiments established:

  1. large MW
  2. extended chain
  3. highly ordered structure
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18
Q

1945 Astbury

A
  • proposed a single chain molecule
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19
Q

1950 Chargaff

A

disproved tetranucleotide [A]=[T]=[C]=[G],

actually [A+G]=[T+C] or purines = pyrimidines

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20
Q

1952 Pauling

A
  • helix with bases on the outside
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21
Q

Cavendish laboratory at Cambridge Univ.

Astbury -

A 
primative x-ray diffraction
1.  bases stacked
2.  sugars in same plane as bases
3.  single chain
4.  internucleotide spacing 3.4 Å, 20 layers complete turn 20 Å
          diameter
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22
Q

Maurice Wilkins and Rosalind Franklin

A

high quality photos x-ray diffraction

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23
Q

Watson and Crick

A

own x-ray work - saw Franklin’s photos

model building

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24
Q

Watson and Crick Proposals

A
  1. double helix (density determinations)
  2. hydrogen bonds between AT GC
  3. antiparallel chain structure (sugars 3’ or 5’ on chain)
  4. worked out a method of replication
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25
DNA STRUCTURE
a. Purines/pyrimidines b. Sugars (differences) c. Backbone d. Complimentary base pairing
26
DNA STRUCTURE -bonds
a. Attachment N H (of base to sugar) b. Ester bond or phosphdiester bond (phosphate in backbone 5') c. N-glycosylic bond, base to sugar
27
Nucleoside
base linked to sugar
28
Nucleotide
base, sugar + PO4
29
Polynucleotide
several nucleotides
30
3' ______ terminusand 5'_______ terminus
OH terminus | -PO4 terminus
31
Two forces that hold DS-DNA together
1. H bonds - base pairing | 2. Hydrophobic interactions
32
Antiparallel 3'OH - 5'PO4
1. each type of terminus at each end allows enzyme activity at either end 2. strand orientation - different replication implications
33
DNA ___ _____ structure
Double helix
34
space models __, ___, ___ forms
B, C, Z
35
Forms of the Double Helix
majority are right-handed helices (natural forms)
36
B formm
92% relative humidity, low ionic strength major groove-protein contact minor grove - Diameter 20 A Watson & Crick model, standard form in organisms 10.4 bp/turn, in vivo cell form
37
A form
75% R.H., counter ions (sodium, cesium) 11 bp/turn, wider diameter major groove much smaller (proteins inaccessible) very close to DS-RNA (2' hydroxyl group causes physical constraints)
38
Z form
left handed helix (in vitro --> in vivo) most bp/turn 12, least twisted diameter - 18 Å name from Z form of backbone only single groove, greater density negative charges alternating purines & pyrimidines (CGCG...) but not always possible in vivo conversion from B --> Z, B
39
Drawing Conventions 5'P Terminus ____ 3' OH terminus ___
left | Right
40
Drawing Conventions
1. ATC 2. p-5'-ATC-3'OH 3. pApTpC
41
Variation of Base Composition
G+C content or percent G+C ([G] + [C]) ________ [all bases]
42
Variation of base composition stats
``` higher organisms ∼ 50% (euk.) lower organisms - wide variation Clostridium .27 Sarcinia .76 E. coli .5 ```
43
Fragility of DNA Molecules
very susceptible to shearing forces (pipeting, mixing, vortexing) if MW less than 2x108 - OK (whole DNA - virus, phage) bacterial DNA - fragmented ∼ 100 pieces mean size - 2.5x107
44
native state
ordered state of a molecule commonly found in nature
45
denatured state
disrupted, random coil
46
denaturation
transition from native --> denatured (hydrogen bonds, hydrophobic bonds) heated
47
DS - DNA (native DNA) --------->
bonding forces disrupted
48
definition: SS DNA =
denatured DNA
49
Melting curves
1. A260 - DNA stable at higher temps then normally found in natural environment 2. 6-8oC denaturation is total in this range 3. rise is about 37% increase
50
Tm
melting temperature - temperature at which the rise is half complete illustrate on graph - always set for organism etc.
51
G-C content vs Tm
higher the GC/higher the Tm
52
Base Stacking Considerations
1. linear DS DNA has frayed ends, 7 bp broken 2. short DS molecules have a low Tm, 3 bp not stable 3. short paired regions - can't maintain at physiological temperatures.
53
Denaturation Methods
1. helix-destabilizing proteins (relaxation, melting proteins) ex. 32-protein E. coli T4 phage used for replication - produced by all cells 2. Alkali conditions pH 11.3 or higher eliminates H-bonds best because heat breaks phosphodiester bonds 3. heat
54
after denaturing:
vary salt conc. to keep the strands separate | low salt + room temp - keep strands from coiling
55
Renaturation
denaturated DNA --> native form
56
Renaturation Uses
1. determine genetic relatedness between DNA species 2. detect RNA species RNA/DNA hybridization 3. repeating sequences detected - copies of a particular gene 4. locate specific base sequences
57
Conditions for Renaturation
1. high salt conc. so PO4 on strands won't repulse (.15 to .5 m NaCl) 2. temp must be high enough to disrupt H-bonds randomly formed - 20-25oC below Tm slow process - random collision of bases once a few bases lined up - zip up quickly bind by backbone- bases stick up
58
Filter Hybridization
radioactive DNA or RNA proberadioactive DNA or RNA probe
59
DNA Heteroduplexes
reannealing or renaturation and electron microscopy to search for deletions and mutations "loop" or "bubble" - shows on electron micrograph
60
Circular and Superhelical DNA
1. intact DNA for most bacteria/viruses is circular | a. Covalently Closed Circle - 2 unbroken complimentary strands, nicked circle-interruption (nick) in 1 strand.
61
superhelix or supercoil:
join a linear molecule - twist one end 360o get one crossover pt or node to relieve strain, 720o then 2 crossovers
62
DNA wants to maintain _____ with ___ bp/turn
right handed (positive), 10
63
all native superhelical DNA are______, therefore have _______ supercoils
underwound, left-handed (negative)
64
Supercoiling
can occur only in closed structures, molecule that lacks supercoiling is relaxed (usually nicked on 1 strand)
65
Fluctuation in base pairing
base pairing, H-bonds are breaking and reforming, DS regions --> SS bubbles
66
Breathing
(occurs naturally) - allows proteins to enter --> occurs in AT rich regions more often
67
Negative supercoils
-allow DNA to relieve torsional pressure -reduces rotation per base pair (loosens the winding of the 2 strands) so DNA is underwound when change in structure of DNA is needed, less energy is required if it is neg. supercoiled supercoiled DNA undergoes structural transitions, relaxed DNA may not undergo (strand separation) *all genomes examined have some neg supercoiling
68
Superhelix density or degree of twisting
1 negative twist/200 bp
69
Origin of supercoiling
``` DNA gyrase or topoisomerase - modify superhelical structure - covered during replication eukaryotes - underwinding is result of DNA wound around histones ```
70
Special Base Sequences - Structural Consequences
- common in regulatory regions, areas of enzyme activity | - impart other special properties (Pu/Pyr - left handed helices)
71
Special Base Sequences | Palindrome
reads the same either way - also called inverted repeat - inverted regions separated by spacer can assume other structures because of breathing
72
RNA
ribose sugar, uracil not thymine 1. typical cell has 10X as much RNA as DNA - why so much? Large # of roles. 2. single-stranded (viruses can have DS RNA) 3. types ribosomal RNA (3-4 forms) rRNA transfer RNA (50 forms) tRNA messenger RNA (1000's of forms) mRNA 4. about 1/2-2/3 of a RNA molecule actually paired a. stem & loop b. hairpin
73
Hydrolysis of Nucleic Acids
breaking apart backbone
74
Hydrolysis of Nucleic Acids (4)
1. low pH (1) phosphodiester bonds broke + N-glycosidic bonds (sugar & base) both DNA & RNA - study base sequence - free bases produced 2. pH 4, N-glycosylic bonds for purines (A,G) broken apurinic acid - all purines are removed 3. heat removes methylated bases - CH3 (add - CH3 to A,G,C, first) 4. pH 11 - RNA totally hydrolyzed to ribonucleotides pH 13 - DNA resistant (37oC) very difference 2'-OH on sugar
75
Nucleases
- enzymes that depolymerize nucleic acids
76
Types of Nucleases
1. DNase or RNase 2. Cut DS or SS conformation (some work on both) 3. site of reaction a. exonucleases b. endonucleases c. ribozymes
77
exonucleases
act only on the termini, specific for 3' or 5' end
78
endonucleases
act within the strand, some at particular base sequences
79
ribozymes
RNA molecules with nucleolytic activity - cleave at specific phosphodiester bonds
80
Eukaryotic cells
– nucleus | Diploid (2 copies) or polyploidy (multiple copies
81
Prokaryotic cells
– nucleoid Haploid (1 copy) Plasmids often polyploidy with multiple copies
82
Genome vs. plasmids
essential cell functions vs. genes not required for growth
83
Most bacteria have only ____ genome
one
84
Bacterial genome is generally _____ eukaryotic genomes are ______
circular, linear
85
Circular chromosomes require
topoisomerases
86
topoisomerase purpose
separate daughter molecules (catenated molecules)
87
No telomeres are required for _____ ______ to replicate
circular genomes
88
Eukaryotic DNA is wrapped around
histones
89
Bacterial genomes have proteins __, __, __, ___ which DNA can be wrapped around
HU, HN-S, Fis, IHF
90
Sequence Motifs in Bacterial Genomes
1. oriC region 2. dif site – 3. ter sites 4. translocase sites 5. chi sites – 6. CTG sequence – 7. Sequences involved in chromosome segregation during sporulation
91
oriC region
initiation of replication
92
dif site
resolution of dimer chromosomes
93
ter sites
– termination sequences in some bacteria, often redundant (multiple copies)
94
translocase sites
– chromosome segregation
95
chi sites
guide recombination proteins (involved in repair of DS-DNA breaks)
96
CTG sequence
priming lagging DNA strand

Microbial Genetics (4 decks)

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