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Microbial Genetics > History of DNA > Flashcards

History of DNA Flashcards

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
Q

DNA STRUCTURE

A

a. Purines/pyrimidines
b. Sugars (differences)
c. Backbone
d. Complimentary base pairing

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26
Q

DNA STRUCTURE -bonds

A

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

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27
Q

Nucleoside

A

base linked to sugar

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28
Q

Nucleotide

A

base, sugar + PO4

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29
Q

Polynucleotide

A

several nucleotides

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30
Q

3’ ______ terminusand 5’_______ terminus

A

OH terminus

-PO4 terminus

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31
Q

Two forces that hold DS-DNA together

A
  1. H bonds - base pairing

2. Hydrophobic interactions

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32
Q

Antiparallel 3’OH - 5’PO4

A
  1. each type of terminus at each end allows enzyme activity at either end
  2. strand orientation - different replication implications
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33
Q

DNA ___ _____ structure

A

Double helix

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34
Q

space models __, ___, ___ forms

A

B, C, Z

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35
Q

Forms of the Double Helix

A

majority are right-handed helices (natural forms)

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36
Q

B formm

A

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

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37
Q

A form

A

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)

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38
Q

Z form

A

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

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39
Q

Drawing Conventions
5’P Terminus ____
3’ OH terminus ___

A

left

Right

40
Q

Drawing Conventions

A
  1. ATC
  2. p-5’-ATC-3’OH
  3. pApTpC
41
Q

Variation of Base Composition

A

G+C content or percent G+C
([G] + [C])
________
[all bases]

42
Q

Variation of base composition stats

A 
higher organisms ∼ 50% (euk.)
lower organisms - wide variation
Clostridium .27
Sarcinia .76
E. coli .5
43
Q

Fragility of DNA Molecules

A

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
Q

native state

A

ordered state of a molecule commonly found in nature

45
Q

denatured state

A

disrupted, random coil

46
Q

denaturation

A

transition from native –> denatured (hydrogen bonds, hydrophobic bonds) heated

47
Q

DS - DNA (native DNA) ———>

A

bonding forces disrupted

48
Q

definition: SS DNA =

A

denatured DNA

49
Q

Melting curves

A
  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
Q

Tm

A

melting temperature - temperature at which the rise is half complete
illustrate on graph - always set for organism etc.

51
Q

G-C content vs Tm

A

higher the GC/higher the Tm

52
Q

Base Stacking Considerations

A
  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
Q

Denaturation Methods

A
  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
Q

after denaturing:

A

vary salt conc. to keep the strands separate

low salt + room temp - keep strands from coiling

55
Q

Renaturation

A

denaturated DNA –> native form

56
Q

Renaturation Uses

A
  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
Q

Conditions for Renaturation

A
  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
Q

Filter Hybridization

A

radioactive DNA or RNA proberadioactive DNA or RNA probe

59
Q

DNA Heteroduplexes

A

reannealing or renaturation and electron microscopy to search for deletions and mutations “loop” or “bubble” - shows on electron micrograph

60
Q

Circular and Superhelical DNA

A
  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
Q

superhelix or supercoil:

A

join a linear molecule - twist one end 360o get one crossover pt or node to relieve strain, 720o then 2 crossovers

62
Q

DNA wants to maintain _____ with ___ bp/turn

A

right handed (positive), 10

63
Q

all native superhelical DNA are______, therefore have _______ supercoils

A

underwound, left-handed (negative)

64
Q

Supercoiling

A

can occur only in closed structures, molecule that lacks supercoiling is relaxed (usually nicked on 1 strand)

65
Q

Fluctuation in base pairing

A

base pairing, H-bonds are breaking and reforming, DS regions –> SS bubbles

66
Q

Breathing

A

(occurs naturally) - allows proteins to enter –> occurs in AT rich regions more often

67
Q

Negative supercoils

A

-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
Q

Superhelix density or degree of twisting

A

1 negative twist/200 bp

69
Q

Origin of supercoiling

A 
DNA gyrase or topoisomerase
- modify superhelical structure 
- covered during replication
eukaryotes
 - underwinding is result of DNA wound around histones
70
Q

Special Base Sequences - Structural Consequences

A
  • common in regulatory regions, areas of enzyme activity

- impart other special properties (Pu/Pyr - left handed helices)

71
Q

Special Base Sequences

Palindrome

A

reads the same either way

  • also called inverted repeat
  • inverted regions separated by spacer can assume other structures because of breathing
72
Q

RNA

A

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
Q

Hydrolysis of Nucleic Acids

A

breaking apart backbone

74
Q

Hydrolysis of Nucleic Acids (4)

A
  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
Q

Nucleases

A
  • enzymes that depolymerize nucleic acids
76
Q

Types of Nucleases

A
  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
Q

exonucleases

A

act only on the termini, specific for 3’ or 5’ end

78
Q

endonucleases

A

act within the strand, some at particular base sequences

79
Q

ribozymes

A

RNA molecules with nucleolytic activity - cleave at specific phosphodiester bonds

80
Q

Eukaryotic cells

A

– nucleus

Diploid (2 copies) or polyploidy (multiple copies

81
Q

Prokaryotic cells

A

– nucleoid
Haploid (1 copy)
Plasmids often polyploidy with multiple copies

82
Q

Genome vs. plasmids

A

essential cell functions vs. genes not required for growth

83
Q

Most bacteria have only ____ genome

84
Q

Bacterial genome is generally _____ eukaryotic genomes are ______

A

circular, linear

85
Q

Circular chromosomes require

A

topoisomerases

86
Q

topoisomerase purpose

A

separate daughter molecules (catenated molecules)

87
Q

No telomeres are required for _____ ______ to replicate

A

circular genomes

88
Q

Eukaryotic DNA is wrapped around

89
Q

Bacterial genomes have proteins __, __, __, ___ which DNA can be wrapped around

A

HU, HN-S, Fis, IHF

90
Q

Sequence Motifs in Bacterial Genomes

A
  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
Q

oriC region

A

initiation of replication

92
Q

dif site

A

resolution of dimer chromosomes

93
Q

ter sites

A

– termination sequences in some bacteria, often redundant (multiple copies)

94
Q

translocase sites

A

– chromosome segregation

95
Q

chi sites

A

guide recombination proteins (involved in repair of DS-DNA breaks)

96
Q

CTG sequence

A

priming lagging DNA strand

Microbial Genetics (4 decks)

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