Molecular Genetics 1-6 Flashcards
What and when was the Hershey-Chase experiment?
1952
Involved the use of a phage (virus) which affects E. coli. The phage could replicate once inside E. coli cells. In one experiment they labelled the protein with radioactive sulphur, and in the other they labelled the DNA with radioactive phosphorus. Then they allowed the phage to infect E. coli and centrifuged the E. coli cells. When the protein was labelled, radioactivity was only found in the liquid, not pellet. Radioactivity was found in both when DNA was labelled
When did Watson and Crick propose the structure of DNA?
1953
Who helped Watson and Crick come to their conclusions on DNA structure?
Rosalind Franklin and PhD student Raymond Gosling
How are the carbons labelled in a DNA molecule?
5’ carbon at phosphate end
3’ carbon has hydroxide group
Which nucleotide bases are purines?
Adenine and guanine
Which nucleotide bases are pyrimidines?
Cytosine and thymine
What is a gene?
The basic unit of inheritance by which hereditary characteristics are transmitted from parent to offspring
What is a gene at the molecular level?
A length of DNA (or in some viruses RNA) which exerts its influence on the organism’s form and function by encoding and directing the synthesis of a protein, or a tRNA, rRNA or other structural protein.
In eukaryotes, a gene also includes noncoding introns
What is the name for the type of mRNA produced in prokaryotic transcription and what does it mean?
Polycistronic
A single strand of mRNA that encodes several proteins
What is the purpose of the 5’ cap of mRNA?
The cap plays a role in the ribosomal recognition of messenger RNA during translation into a protein
What enzyme places a 5’ cap on the end of the mRNA molecule?
Gaunyl transferase
What is the poly A tail at the 3’ end of the mRNA molecule?
It is a string of adenine bases. It’s addition is catalysed by the enzyme poly (A) polymerase
Where is DNA found? (4 places)
Nucleus
Cytoplasm
Plastid
eDNA (environmental DNA)
In what direction is mRNA transcribed?
In a 5’ to 3’ direction along the coding strand, against the direction of the template strand
What is eDNA?
Found in soils, water, faeces, etc
Useful for ecological surveying - what species are in the area
DNA in our food can test if packaging is honest about contents
How is prokaryotic DNA packaged?
- Arranged into nucleoids
- Supercoiled with the help of architectural proteins
- Usually circular DNA genome with nonessential genes in plasmids
- Eukaryotic organelles resemble prokaryotes in their genomic organisation
How is eukaryotic DNA packaged?
- Packaged into linear chromosomes
- Chromatin - DNA supercoiled using histones
- Nucleus is membrane-bound
- Some eukaryotic genomes organised into operons
How are chloroplasts inherited?
Through the cytoplasm - maternally inherited
Steps of plasmid inheritance
- F plasmid carries genes necessary for conjugation
- Plasmid may have additional genetic information from chromosome
- One strand of F+ cell plasmid DNA breaks at arrowhead
- Broken strand peels off and enters F- cell
- Donor and recipient cells synthesise complementary DNA strands
- Recipient cell now also has copy of F plasmid
Prokaryotic DNA replication
- The origin of replication (ORI) is where replication begins
- Replication forks in both directions form, leaving two daughter molecules
Eukaryotic DNA replication
- Multiple ORIs, multiple replication forks and bubbles
2. Two daughter DNA molecules formed
Role of topoisomerase in DNA replication
Breaks, swivels and rejoins the parental DNA, ahead of the replication fork, relieving the strain caused by unwinding
What is the role of helicase in DNA replication?
Unwinds and separates the parental DNA strands
What is the role of primase in DNA replication?
Synthesises RNA primers, using the parental DNA as a template
What is the role of DNA polymerase I in DNA replication?
Removes the RNA primer and synthesises DNA fragments in its place
What is the role of DNA polymerase III in DNA replication?
Synthesises new DNA strand in 5’ to 3’ direction
What is the role of single-strand binding proteins in DNA replication?
Stabilise the unwound parental strands
Which DNA strand is easy to replicate?
The leading strand, as this strand is being replicated in the 5’ to 3’ direction
Which strand is harder to replicate and why?
The lagging strand is harder to replicate as it runs in 3’ to 5’ direction. Primase must produce many RNA primers and hop along placing primers periodically along the DNA
What are the four main steps of DNA extraction?
- Cell lysis and protein removal
- Precipitation
- Wash
- Resuspension (or ELUTION)
Cell lysis - why do you need it?
Lysis of the cell membrane is required to release the DNA
What is in a cell lysis buffer?
- Detergent (e.g. SDS, an an ionic surfactant) - to help break up fats and open up cells
- Salt (e.g. NaCl) - to help break down hydrogen bonds between DNA strands, enabling it to dissolve in the solvent
- EDTA - chelation of divalent ions, which prevents DNases from working
- Tris - acts as a buffer to stabilise alkaline pH
Why would you want to remove proteins during DNA extraction?
DNA is packaged in proteins, so this must be removed. You may also want to remove any contaminating cellular proteins
What happens to proteins when exposed to the lysis buffer?
They are denatured and dissolved by the lysis buffer
What other chemicals assist in protein removal?
- Proteinase (e.g. proteinase K)
- Chaotropic salts (e.g. guanidine hydrochloride) - weaken the forces holding the protein together, thus leading to denaturation
- Ammonium acetate - this will cause proteins to precipitate, so you can centrifuge the solution and remove unwanted proteins which will form pellet
What is precipitation in DNA extraction?
Separating the DNA from the proteins by DNA precipitation
What is added to precipitate the DNA and how is it physically separated?
Isopropanol or ethanol - DNA is insoluble in both of these chemicals, so it leads to the aggregation of DNA molecules out of solution. DNA can then be separated by centrifugation or with a silica matrix in a column
Why must the DNA pellet or matrix be washed?
To remove contaminants
How is the pellet washed?
With 70% ethanol - the salts you are trying to remove will not dissolve in 100% ethanol
How is resuspension carried out?
DNA is soluble in water, so most DNA preps will use purified water to re-suspend the pellet of DNA or to pass through the matrix and collect the DNA elute in a tube.
Another popular resuspension solution is TE buffer, which includes Tris and EDTA
What is in vivo DNA synthesis?
DNA replication within a cell
What is in vitro DNA synthesis?
DNA produced in a test tube/lab
What are the building blocks for synthesising DNA in nature?
Deoxyribonucleoside triphosphates (dNTPs). As the new base gets added to the 3’ end of the chain, two of the phosphates are lost
What are used instead of dNTPs when building DNA synthetically (without the use of enzymes)?
Phosphoramidites