Cell Biology- Hejmadi lecture 1+2

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When, who and how were nucleic acids discovered?
1869. F Meischer- pus filled bandages put in a light salt solution and alkaline added- pus cells burst to produce white ppt- something inside cells!
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When, who and how was the transforming principle discovered?
F. Griffith in 1928- by experimenting with Streptococcus pneumoniae, a bacteria of which there are two strains which will infect mice.
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What are the two strains?
a)smooth strain (virulent) -protective capsule protects it from host’s immune system (mouse will die if injected with it) b) rough strain (non-virulent) -no protective capsule- defeated by host’s immune system (mouse survives if injected with it)
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What happens if the virulent strain is heated and then injected into mouse? What does this show?
Mouse survives- virulence is lost due to heating
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What happens if the virulent strain is heated and mixed with a live non-virulent strain then injected into the mouse? Why?
The mouse dies. demonstrates how some DNA from heat-killed smooth strain has been taken up by live rough strain, converting it into a virulent strain.
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When and who discovered that transforming principle was a nucleic acid called DNA, which hence is genetic material.
1944- Avery, McCleod & McCarty
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How did they discover this? (2 steps- 1st step on this slide)
They started with a mixture of heat-killed virulent strains with living non-virulent strains- which should result in S form of the bacteria if transformation is able to occur.
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Step 2
2) They then eliminated different components of the cell one at a time by systematically adding different enzymes until there was no transformation
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What did they find?
When DNA was destroyed by DNase, there was no transformation of the non-virulent strain (remained in R form), hence demonstrating that DNA must be the genetic material in a cell.
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When and who found the base ratio?
1953- Erwin Chagraff
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What is the base ratio?
amount of A=T amount of C=G. He discovered that the % of each of these remained constant throughout a population of a given species, but varied between species. i.e. chagraff’s ratio is universal – only % A-T pairs with C-G pairs varies
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What did Hersey and Chase confirm in 1952? How?
That DNA was the genetic material- through the 'blender' experiment
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What did they conduct their experiment on?
a virus known as T2 phage, which consists of a protein shell containing its genetic material.
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How does the phage infect a bacterium?
by attaching to its outer membrane and injecting its genetic material and leaving its empty shell attached to the bacterium.
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Describe the 2 experiments they carried out. (first experiment)
1)The DNA of phages was labelled with radioactive P32. They allowed the phages to infect E. coli, and observed the transfer of P32 labelled phage DNA into the cytoplasm of the bacterium. 2)
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Second experiment
2)The phages were labelled with radioactive 35S. Following infection of E. coli they then sheared the viral protein shells off of infected cells using a high-speed blender and separated the cells and viral coats by using a centrifuge.
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What did they observe from the second experiment?
After separation, the radioactive S35 tracer was observed in the protein shells, but not in the infected bacteria
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Why was this the case?
phosphorus is present in DNA but not in any of the 20 amino acids from which proteins are made, whilst Sulfur is present in the amino acids cysteine and methionine, but not in DNA
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What else happened in 1952?
R Franklin & M Wilkins- DNA diffraction patterns using crystallography
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What did this allow?
dimensions could be calculated.
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Why was their X-ray diffraction pattern clearer than others?
because water was included in the DNA sample
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What happened in 1953?
J Watson & F Crick proposed their double helix model of DNA structure
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What did Linus Pauling incorrectly suggest about the structure of DNA?
a triple helix in which phosphates formed the helical core with bases pointing outwards.
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Why was it incorrect?
It would be impossible under normal cellular conditions as each phosphate group is negatively charged, and hence if forced together in a helix would repel eachother- literally driving the structure apart.
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What 3 components are nucleotides made up of?
1. 5 carbon sugar (pentose) 2. Nitrogenous base (A,T, C or G) 3. Phosphate group
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What are the 2 key differences between RNA and DNA?
1) the lack of an oxygen on the 2’ carbon on the sugar in DNA 2)Uracil present in RNA in the place of thymine in DNA.
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Ensure you can draw general structure of DNA and RNA AND all the 5 nitrogenous bases ...
...(check that you can in cell biology lecture 1 p.4-5)
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What is a pyramidine?
A base with a single aromatic chain (thymine, uracil and cytosine)
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What is a purine?
A base with a double aromatic chain (adenine and guanine)
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How does the structure of purines and pyramidines help to explain Chagraff's ratio?
We know that DNA has a uniform width of 20 A diameter, which means by process of elimination purines MUST always pair with pyrimidines
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How many hydrogen bonds are there between... a) A and U/T b) C and G
a) 2 b) 3
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What is a nucleotide?
phosphate ester monomer of pentose
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What is an oligonucleotide?
short polymer
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What is a polynucleotide?
long polymer >10 nucleotides long
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What is a nucleoside?
monomer of sugar + base (no phosphate)
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What 2 types of bonds hold the DNA molecule together? (not H bonds)
A)N-glycosidic bonds -link nitrogenous base to 1’C in pentose sugar in beta configuration B)Phosphodiester bonds -gives the backbone to the polymer -phosphate group linked to 5’C of one pentose sugar, and to hydroxyl on 3’C of another
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Draw the structure of 2 nucelotides linked together and label these 2 particular bonds
Look at cell biology lecture 1 p.6 for reference
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What is the direction of polarity in a DNA molecule?
5'-->3'
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What are the three main DNA conformations?
A, B and Z
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What is the standard conformation that we are used to seeing? Give 9 of its key features. (first 8 on this slide)
•right twisting •double helix • uniform 20A diameter •3.4nm from one turn to the next •0.34nm between each base •minor grooves between two minor turns •major grooves created by two consecutive turns of the DNA •anti-parallel strands
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9th feature
• bases on inside perpendicular to axis
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You should be able to compare the 3 conformations on....
...Orientation of helix, Width Inclination of base pairs, Central axis ,Major groove, Minor groove, Side view, Top view
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Give 3 structural differences between DNA and RNA
RNA: intra-molecular base pairing leads to structures such as hairpin loops, single stranded – yet still does twist to form secondary conformation -less pronounced major and minor grooves
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Give 4 classes of RNA and what their function is
1)Transfer RNA- mRNA binding, involved in protein synthesis 2)Ribosomal RNA- ribosomal subunits involved in protein synthesis 3)snRNA- RNA splicing- cuts out introns 4)Small nucleolar RNA (snoRNA)-rRNA modification and processing- found in nucleolus
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PROPERTIES OF DNA LECTURE 2
....
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What is the c-value of an organism?
the entire haploid DNA content of any cell i.e. the DNA content in one chromosome from any cell.
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Why is genome size not linked to genetic complexity?
due to the presence of repetitive (junk) DNA- only 2% of human DNA is coding. It is how proteins come together and function that gives an organism complexity.
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How does protein domain contribute to complexity?
-number of exons that are needed to make a protein can vary hugely -folding -interactions -side chains added -function etc.
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Why is supercoiling topology important?
It means a large amount of DNA can fit into a very small nucleus
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Why do we need histones?
DNA is a negatively charged molecule due to its phosphate groups- so need histones in order for it to compress
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What are histones?
positively charged octimers- made up of 8 subunits of predominantly positively charged amino acids (it’s a protein)
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What do they do?
neutralise the negatively charged phosphate groups - therefore DNA winds around histones in order to compress to form chromatin
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What are the two ways for DNA to undergo supercoiling?
1)negative supercoiling 2)positive supercoiling
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Describe negative supercoiling
results from under/unwinding -important in DNA packing/unpacking e.g. during replication/transcription
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Describe positive supercoiling
-results from overwinding -also packs DNA but more difficult to unwind
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How does supercoiling relate to speed of DNA migration?
Most supercoiled= faster moving
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What 2 forms does DNA supercoiling take?
toroidal (DNA wound around histones) and interwound (- DNA winding around itself - no histones involved)
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What 2 enzymes are involved in the unwinding process?
Topoisomerase I- single strand nicks and Topoisomerase II- double strand breaks
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Describe how topoisomerase I acts
Cuts one strand of DNA by breaking the backbone through breaking the phosphodiester link via tyrosine active site. This transfers the diester link onto itself.
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Describe how topoisomerase II acts
This cuts both strands of DNA typically at the same site. This is an energy driven process and hence requires ATP
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Give an example of topoisomerase II's importance
replication of bacterial DNA - At time of replication we will get an interwound ring- the two newly replicated DNA will be wound around eachother. Must separate to go into each daughter cell
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What do antibiotics/chemotherapeutic agents do?
inhibit the activity of these enzymes. Hence DNA remains coiled together or doesn’t fully separate leading to cell death
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Describe denaturation of DNA. What else is this known as?
occurs abruptly when DNA is heated to a certain temperature. It occurs within a very narrow temperature range known as Tm. Also known as melting.
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What is formed from the denaturation?
a fluctuating conformational state (‘random coil’), which results due to the breakdown of hydrogen bonds holding the two strands together.
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What is Tm?
the temperature at which DNA is half double stranded, half single stranded.
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Why does DNA absorb Uv light at 260nm?
Due to its nitrogenous bases
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What is hyperchromicity?
the increase in absorbance of a material
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Single stranded DNA absorbs ___% more UV light than double stranded DNA as ..... What does this imply?
37%, as the bases become unstacked. Therefore from DNA melting curve we can find Tm to be where absorbance of UV light is 50% between maximum and minimum!
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Why does Tm increase with GC content?
This is because it requires more energy to break the 3 hydrogen bonds between GC than the 2 hydrogen bonds between AT, hence meaning GC rich DNA will denature at a higher temperature, and hence Tm will be higher
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Describe annealing (renaturation)
when the two separate denatured DNA strands join together to form once again double stranded DNA.
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At what temperature in relation to Tm does annealing occur?
typically 25C below Tm
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Front

When, who and how was the transforming principle discovered?

Back

F. Griffith in 1928- by experimenting with Streptococcus pneumoniae, a bacteria of which there are two strains which will infect mice.

Card 3

Front

What are the two strains?

Back

Preview of the front of card 3

Card 4

Front

What happens if the virulent strain is heated and then injected into mouse? What does this show?

Back

Preview of the front of card 4

Card 5

Front

What happens if the virulent strain is heated and mixed with a live non-virulent strain then injected into the mouse? Why?

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