1
Q
Do eukaryotes or prokaryotes contain histones? What are histones?
A
- Histones: basic (alkaline) and positively charged proteins
- Only eukaryotes contain histones, prokaryotes don’t have histones and therefore have ‘naked’ DNA
2
Q
What is a nucleosome, what are its functions and what is the link to eukaryotic structures?
A
- A length of DNA of 150 base pairs, wrapped around eight histones (four pairs of four different histones) and H1 (type of histone)
- DNA is acidic and negatively charged whereas histones are basic, the bonding neutralizes the DNA
- DNA strand flows from each nucleosome to the next, hence linking them
- The DNA that links them is called DNA linker
- This is called string of beads (DNA and histones together)
- Nucleosomes help supercoil the DNA while ensuring appropriate access to it
- When the coils in DNA unwind by helicase, DNA copies can be made and nucleosomes get access to the DNA
- Nucleosomes are the repeat units of eukaryotic chromatin (later eukaryotic chromosomes)
Diagram in book
3
Q
What does base paring do? How many hydrogen bonds exist between which bases?
A
- DNA replication relies on base pairing which allows the stability of the double helix
- This is because the hydrogen bonding between the purine and the pyrimidines
- Double hydrogen bonds exists between adenine and thymine
- Triple hydrogen bonds exist between guanine and cytosine
- T has a slightly positive charge and A a slightly negative charge which allows the bonding of the two bases
4
Q
What bases are bonded together?
A
- Pyrimidine and purine
- Purines: Guanine and Adenine (contain two rings in their structures)
- Pyrimidines: Thymine and Cytosine (contain one ring in their structure)
5
Q
How does DNA replication differ between eukaryotes and prokaryotes?
A
- In eukaryotes replication is initiated at various points along the DNA
- Rate of replication is 100 nucleotides per second
- In prokaryotes replication is started at only one position along the DNA
- Rate of replication is 1000 nucleotides per second
- The DNA replication is more efficient in prokaryotes
- You only need to know the prokaryotic process
6
Q
What 7 enzymes and their functions are involved in DNA replication?
A
- The process is carried out by a complex system of enzymes
- Helicase: unwinds and unzips DNA by breaking the hydrogen bonds, causes supercoiling
- DNA gyrase: takes over helicase’s function. It relieves strain on the strand outside the replication fork
- Single-stranded binding proteins: bind to new strand formed to keep the original strands apart, prevent supercoiling
- DNA primase: adds primers to the DNA strands
- DNA ligase: adds missing phosphate on lagging strand
- DNA polymerase I: removes primers and adds DNA segments, found only on the lagging strand
- DNA polymerase III: duplication of the DNA on both the lagging and leading strand and is found at the replication fork. Adds nucleotides to the 3’ end of the RNA primers
7
Q
When do the enzymes work during DNA replication?
A
- Helicase unwinds the DNA double helix first, then the single-stranded binding proteins bind to the single strands formed so that the DNA sequence can be copied
- Helicase causes supercoiling which is then taken over by DNA gyrase
- Nucleoside triphosphates bind to the template, by which they lose their two extra phosphate groups to generate energy
- DNA polymerase III adds DNA nucleotides to the strands but only to the 3’ OH group of the deoxyribose. It follows the leading strand.
- An RNA primer, found on the leading strand, helps DNA polymerase create a new DNA
8
Q
What role does DNA polymerase III have on the lagging strand?
A
- Because DNA polymerase III can only add a nucleotide to the 3’ OH group (on the leading strand), on the lagging strand the last nucleotide ends with a 5’ phosphate group
- On the lagging strand, the DNA primase makes short RNA primers which allow the DNA polymerase III to add DNA nucleotides to the 3’ OH of the RNA primer
- A lot of these primers are required.
- On the lagging strand, short fragments called Okazaki fragments are left due which contain RNA primer and DNA fragment
- DNA ligase joins the fragments together to form a complete DNA strand
- The result are two new strands both based on the template of the old DNA
- DNA replication always progresses from 5’ to 3’ as DNA polymerase III can only add nucleotides to the 3’ OH
9
Q
What was the Rosalind Franklin and Maurice Wilkins DNA structure?
A
- They worked on X-ray diffraction of DNA
- Franklin was able to analyse the DNA crystals
- X-ray diffraction is based on the principle that X-rays are scattered when they pass through different material. Patterns in the diffraction (scattering) indicate properties of the crystals.
- Franklin made a X-ray diffraction radiogram based on the Watson and Crick DNA structure
- The radiogram shows the distance between the base pairs and the turns of the helix
- The distance between the repeats was measured too
- Wilkins showed Franklin’s x-ray data to Watson and Crick to confirm their 3D theory of DNA
10
Q
What was the Hershey-Chase experiment?
A
- Their experiment showed that DNA made up genetic material instead of a protein. They used T2 bacteriophage (virus that infects bacterial cells)
- Radioactive phosphorus and sulfur were used. Phosphorus is found in DNA but not in protein and sulfur is found in protein and not in DNA.
- This way it could be detected which part of the virus entered the bacterium
11
Q
What were the steps of the Hershey and Chase experiment?
A
- Two experiments were done, one with radioactive DNA and one with radioactive protein
Radioactive DNA: - A virus and bacteria were put together and a radioactive DNA (containing phosphorus) enters the bacterium. The radioactivity stays inside the bacterium
- The radioactivity was discovered in the pellet (bottom part), since genetic material contained P, genetic material was present in the bacterium
Radioactive Protein: - A virus and bacteria were put together (containing sulfur) and remains outside the bacterium
- The radioactivity was discovered in the supernatant (top), stating that the virus coat contain sulfur, genetic material does not contain sulfur
12
Q
What were the results and conclusion of the Hershey and Chase experiment?
A
- When bacteriophages containing radioactive phosphorus (32P) infected nonradioactive bacteria, all infected cells became radioactive
- When the bacteria were infected by bacteriophages with radioactive sulfur (35S) and the virus coats removed, almost no radioactivity could be detected in the infected cells
- DNA entered the bacteria whereas the protein component remained outside, the DNA was the genetic material
13
Q
What are non-coding sequences?
A
- Some regions of DNA do not code for proteins, they are found in eukaryotic genomes
- Non-coding DNA accounts for more than 98% of the human genome
- Non-coding DNA: DNA sequences within a genome that do not consist of the information to make a protein
- Non-coding DNA do have other important functions, which is not always known
14
Q
What are four functions of non-coding DNA?
A
- Regulators of gene expression: DNA sequences that regulate gene expression in different ways. Some act as a binding point for RNA polymerase that catalyse the transcription process. Others are called enhancers or silencers that act as binding sites for proteins that increase or decrease the rate of transcription
- Introns: DNA base sequences found within eukaryotic genes that get removed at the end of transcription
- Telomeres: repetitive sequences that protect the ends of chromosomes, they also ensure that DNA is replicated correctly. During cell division short DNA segments are lost from telomeres
- Genes for tRNA’s: these genes code for RNA molecules that do not get translated into proteins but form tRNA which are involved in translation
15
Q
What are tandem repeats?
A
- Tandem repeat: a sequence of two or more DNA base pairs that is repeated so that the repeats lie end-to-end on the chromosome
- Tandems repeats form part of non-coding DNA, however they may be present in protein-coding regions
- The number of repeated DNA segments varies from individual to individual and used for identification in DNA fingerprinting
Check book
16
Q
For what are tandem repeats used for?
A
- For DNA profiling (DNA fingerprinting) which is used to identify individuals by analyzing their DNA
- Tandem repeats vary among individuals, therefore their differences in these regions can be analyzed to produce a DNA profile
- DNA profiling used: crime scene, tests for parentage
17
Q
How does DNA profiling work?
A
Steps:
- Collection of samples and extraction of DNA
- Amplification (copying) of the DNA region containing tandem repeats by PCR
- Separation of the DNA fragments using gel electrophoresis
- Short tandem repeats are used (STRs)
- When two alleles are separated, the allele with the most repeats is separated from the allele with the least repeats
- The differences are seen on the gel. Depending on the position of the alleles, it creates the DNA fingerprint
- Each DNA fragments of an individual must be traced to either one of the parents
Check book
18
Q
What is DNA sequencing and what is it used for?
A
- A method used for deducing the precise order of nucleotides within a DNA molecule. It determines the order of the four bases (adenine, guanine, cytosine and thymine) in a DNA strand
- Allows the precise order of bases in a DNA strand to be determined
- Purposes: DNA profiling, paternity suits, forensics, cancer analysis and genome studies
19
Q
How does DNA sequencing work?
A
- The technique is called Dideoxy Chain Termination Method
- Structures required: DNA templates, primer, DNA polymerase and 4 nucleotides
- DNA polymerase needs a 3’ OH group of the nucleotide to add another nucleotide to the DNA strand
- If a dideoxy nucleotide (lacking 3’ OH group, only a H) is added to the mixture, this nucleotide is built into the growing strand, and no further nucleotides can be added and the reaction stops
- A fluorescent dye to the four dideoxynucleotides is added to recognize the base present after replication stopped
- Because there are so many combinations of ddNTPs, there is a DNA fragment of every possible length and it is known which of the nucleotides is at the end of the fragments
- In the end each nucleotide at every position is known
20
Q
Nucleotides = bases in DNA (A, T, C, G)
A
7. Nucleic acids
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