DNA Flashcards
Traits of a genetic material
replication
storage of information
expression of information
variation by mutation
Tetranucleotide hypothesis
DNA is made of linked, repeating tetramers of nucleotides
all 4 nucleotides present in equal amounts
DNA was therefore uniform and simple
Incorrect
Who discovered DNA and how
Miescher
He found nucleic acids, derived from the nucleus of a WBC, which he called “nuclein”
Nuclein
nucleic acids, derived from the nucleus of a WBC; Miescher’s discovery of DNA
Miescher’s assumptions and discoveries about nuclein
ASSUMPTIONS:
He believed it was novel due to its resistance to protease digestion
DISCOVERIES:
He found it was in all nuclei
Why was protein thought to carry genetic information, as opposed to DNA?
DNA seemed to lack chemical diversity
early tetranucleotide hypothesis stated nucleotides were in short repetitive sequences, so did not seem to encode anything
protein has structural diversity
GRIFFITH Experiment design
two strains of bacteria: one virulent, one avirulent
heated to death some of the bacteria
introduced bacteria (both alive and dead) into mice in varying combinations
1 avirulent, 1 virulent, 1 dead virulent, 1 dead virulent + living avirulent
GRIFFITH results
IIR, living
mouse lived
IIIS, living
mouse died
dead IIIS
mouse lived
dead IIIS and IIR
mouse died
when the dead virulent and living avirulent strain were mixed, the mouse died
living IIIS was recovered from the dead mouse
GRIFFITH conclusions
Something in the dead IIIS must be making the IIR virulent.
He called this the transforming principle.
a transforming principle exists in bacteria which allow them to pass traits to other bacteria
Transforming principle
Something taken up from the environment by one bacterium, which was shed by another, and allows it to transform or change to be like the principle’s source
allows the passage of traits to other bacteria
Griffith’s virulent strain
S FORM (smooth)
encapsulated
look smooth due to capsule
Griffith’s avirulent strain
R FORM (ROUGH)
no capsule
look rough
Serotype
refers to the antigens on the surface of the bacterium
each elicits a different immune response
Serotypes in Griffith’s experiment
In his experiments, Griffith used these two serotypes:
IIR, avirulent
IIIS, virulent
Why is one strain virulent and the other avirulent?
The difference in virulence depends on the presence of a glycocalyx.
The glycocalyx prevents engulfment of the bacteria, making them virulent.
They multiply and cause pneumonia.
IIR
avirulent
rough, no capsule
IIIS
virulent
smooth, encapsulted
AVERY Experiment design
heat-killed IIIS were broken up using detergent
extract of cellular contents was made
TWO DIFFERENT EXTRACTS:
1. protein was removed from extract using protease
2. DNA was removed using enzyme
IIR exposed to extracts to see if they would transform
AVERY results
for the extract with no DNA, no changes occurred in the IIR cells
for the extract with no protein, some IIR cells became IIIS
AVERY conclusions
Once DNA was removed from the filtrate no conversion occurred; so DNA is the transforming principle
HERSHEY & CHASE experiment design
The atoms unique to DNA and protein were labeled in two different groups of viruses using radioactive isotopes.
DNA: P
Protein: S
The labeled phages were allowed to infect bacteria
Cultures blended and centrifuged to produce pellets
shells of phages were separated out with supernatant
HERSHEY & CHASE results
Which material is associated with the pellets? That material is what was injected into the cell.
Radioactive phosphorus — DNA was the pellets
HERSHEY & CHASE conclusion
DNA is the genetic material
Bacteriophage structure
Phages have a protein capsid containing phage DNA. That’s all! Only protein and DNA.
Bacteriophage replication
For the virus to replicate, it must use host cells; so the virus must be injecting its genetic material into a host.
HERSHEY & CHASE purpose
analyzed what a virus was injecting into host cells for the sake of replication (what is the genetic material)?
Transfection experiments
Lysozyme was used to destroy the E. coli cell wall
Purified phage DNA (no proteins) was exposed to the bacteria
no cell wall = easier uptake of DNA
new phages were produced
DNA alone contains all necessary information for the production of phages
Transfection definition
DNA in the environment can be uptaken by other bacteria
Proof of DNA being genetic material in eukaryotes: distribution
DNA is only found where primary genetic function is known to occur; protein is ubiquitous
DNA content corresponds with ploidy level
Proof of DNA being genetic material in eukaryotes: mutations
mutagenesis corresponds to absorption spectrum of DNA, not protein
the more UV light DNA absorbs, the more mutations
Best nm for absorption of UV by DNA
most absorption at 260 nm
QB bacteriophage
RNA is genetic material
has RNA replicase
RNA replicase
Allows creation of more RNA from RNA template
RNA to RNA
Retrovirus
RNA is genetic material
Reverse transcriptase used to make DNA copy of RNA genome
Inserts this DNA copy into your genome
Reverse transcriptase
RNA-dependent DNA polymerase
Usually, from DNA, you make RNA
Now you use RNA to make DNA
RNA to DNA
Which RNA virus has which enzyme?
Retrovirus: Reverse transcriptase
QB bacteriophage: RNA replicase
Nucleic acid
Nucleic acids are polymers
Their monomers are nucleotides
DNA and RNA are nucleic acids
Nucleotides
the monomers of nucleic acids
made of 3 components:
sugar + base + phosphate group
dNMP
nucleoside monophosphates
another word for nucleotides
nucleoside + 1 phosphate group
Nucleoside
sugar + base; an abbreviation of a nucleotide’s components, minus the phosphate
dNTP
nucleoside triphosphates
precursor molecule for DNA synthesis
extra phosphate groups are used as a source of energy for the synthesis process
Two major types of nucleic acid & their difference
DNA and RNA
difference in sugar base: one is missing an oxygen in their hydroxyl group
Purine
A, G
larger, it has 2 rings
Pyrimidine
C, T, U
narrower, it has 1 ring
DNA has a T, RNA has a U
What are incoming monomers to DNA molecules?
nucleoside triphosphate
Where are incoming monomers added to DNA molecules? How?
the 3’ hydroxyl group
nucleoside triphosphate incoming
One phosphate is linked to the hydroxyl group
two phosphates are hydrolysed
breaking bonds provides energy for the exergonic DNA synthesis
sugar-phosphate backbone
the hydroxyls of the sugars, and the phosphate groups attached to the sugars, are what chain nucleotides together
Key features of Watson and Crick’s double-helix model
2 polynucleotide chains aligned
chains wrapped around an axis
sugar-phosphate backbone is on outside
base pairs are “stacked”
grooves exist in the helix
diameter is unchanging
Why is the sugar-phosphate backbone on the outside?
Hydrophilic environment surrounds DNA
Phosphate is charged, so the backbone is hydrophilic itself
This repels unwanted interactions from the bases
Purpose of grooves in helix structure
Allows nucleotides to be partially exposed
Binding sites for various proteins are within the grooves
Minor groove
Groove where the two parts of the backbone are close together
Major groove
Groove where the two parts of the backbone are far apart
5’ end
phosphate group
3’ end
hydroxyl group
Antiparallel
nucleic acids have “ends”
if one end is 3’, it is across from a 5’ end
Allows for complementary base pairing
Complementary base pairing
A & T
G & C
purines and pyrimidines ONLY, otherwise diameter of DNA will change
A&C and G&T don’t bond because those couplings don’t provide maximum number of H-bonds
How do base pairs bond & significance of this
H-bonds
More H-bonds, more stable helix
semi-conservative model of DNA replication
helix unzipped
Single strands have a new daughter strand synthesized by following the base pairs of the original strands
The original strands are conserved
original-daughter helix created
How is RNA different from DNA?
in RNA, sugar is oxygenated
uses uracil in place of thymine
RNA is single strand
snRNA
processes mRNAs for post-transcriptional splicing
Use of miRNA, siRNA, lncRNA
involved in gene regulation
Telomerase function
Extends the DNA ends (telomeres) so that they do not devour themselves in DNA replication
How does telomerase work
There is an RNA sequence inside of it which functions as RNA primer
Melting temperature (tm)
represents point at which 50% of strands are melted
can be used to estimate base composition of DNA
hyperchromic shift
UV absorbance increases as DNA is denatured
when melt DNA, the helix dissociates, and the exposed bases are able to absorb UV
Melting profile
absorption of UV plotted against temp
increases with increasing temp
tm relation to base composition
G-C has more H-bonds than A-T
H-bonds absorb energy when broken
DNA rich in G-C = higher temp needed to melt
Renaturation
DNA reforms h-bonds after being denatured by heat
What are 3 things DNA can renature with?
The original DNA fragments
DNA from different organisms
between DNA and RNA strands
Probes
nucleic acid like DNA or RNA
labeled and can be detected
Used to identify complementary sequences in denatured DNA
Molecular hybridization
DNA renaturation rejoins whatever’s closest and complementary enough; perfect complementation not needed
DNA can be manipulated to rejoin with whatever
FISH stands for what
fluorescent in situ hybridization
FISH concept
mitotic cells are fixed to slides and subjected to hybridization condition
fluorescent ssDNA or RNA is added to detect similar sequences
The added nucleic acids serve as a probe, hybridize only with areas that have sufficient complementarity
added nucleic acids will fluoresce under a microscope, allowing detection
Electrophoresis
Separates DNA and RNA fragments by size in an electric field
DNA and RNA are negative, go towards a positive cathode
small fragments travel faster than large ones
