Molecular DNA Flashcards
What is the C-value paradox?
There is no systematic relationship between genome size and organismal phenotype, although eukaryotic genomes tend to be larger
What are transposable elements?
Pieces of DNA that are able to jump from one chromosomal location to another. The original and new DNA sites are brought together by enzymes and other proteins in a process called transposition
How is DNA replicated?
Using semi-conservative replication - where each strand serves as the template for replication of a new strand of DNA
How many origins of replication are there in bacteria compared eukaryotic genomes?
Bacteria: one origin of replication
Eukaryotes: hundreds or thousands
Enzymes required for DNA replication
Helicases Single-stranded binding proteins Topoisomerase Primase DNA polymerases DNA ligase
Features of DNA polymerase
At least 11 DNA polymerases in eukaryotes
DNA polymerase cannot start a DNA chain, it can only extend it
DNA polymerases add nucleotides in a 5’ -> 3’ direction
What is antiparallel elongation?
Replication takes place in different directions for each complementary strand, due to the antiparallel nature of DNA helices
How do eukaryotes ensure their chromosomes don’t get shorter after each replication?
After replication telomerase adds nucleotide bases to the telomeres at the end of each chromosome
Process of gene transcription in eukaryotes
- RNA polymerase binds to the transcription initiation complex, which is made up of transcription factors and the area of DNA called the promoter
- It separates the 2 strands
- It then moves along the DNA strand, where complementary RNA molecules have now assembled, and joins the RNA nucleotides together
- mRNA is synthesised in the 5’ to 3’ direction
- In eukaryotes, polymerase II is used for mRNA synthesis
What is the 5’ cap in eukaryotic RNA?
A 7-methylguanosine cap added to the 5’ end while elongation is still in progress. This protects the RNA and allows the initiation of translation
What is a poly (A) tail in eukaryotic RNA?
Adenylate residues at the 3’ end, which provide stability
What splices out introns from pre-mRNA and how does it do this?
The spliceosome - a large complex made of proteins and small RNAs
Binds to several short sequences along the intron and releases it
Two flanking exons joined together
What is alternative splicing?
A single gene can encode more than one polypeptide - multi-exon genes can be spliced at least two different ways, depending on TEMPERATURE
What are ribosomes and what do they do?
Perform translation by catalysing the formation of peptide bonds between amino acids
Made of proteins and rRNA
Have a small and large subunit
Initiation of translation
The small and large subunits assemble when attached to an mRNA molecule
The small ribosomal subunit binds to the mRNA
The large subunit completes the translation initiation complex
Termination of translation
The ribosome reaches a stop codon on the mRNA
A release factor binds directly to the stop codon in the A site and promotes hydrolysis
A water molecule is added instead of an amino acid
The ribosomal subunits and other components dissociate
What is a polyribosome?
A complex of mRNA and multiple ribosomes in which several ribosomes are simultaneously translating one mRNA molecule
Why is a start codon so important?
Ensures the gene’s DNA sequence is always read in the correct of the three possible reading frames, therefore coding for the same polypeptide chain
What is the start codon?
AUG
What are the stop codons?
UAG, UAA or UGA
What are operons?
Groups of genes that function as a single transcription unit, so are under the control of the same promoter
What are examples of enzymes that can control transcription factors
Protein kinase - phosphorylate their substrate protein
Protein phosphatases - dephosphorylate their substrate protein
Phosphorylation of transcription factors can control gene transcription
What is another name for a mutation?
Genetic polymorphism
What are examples of small-scale mutations?
Substitutions
Insertions
Deletions (indels)
What are examples of large scale mutations?
Alterations to chromosome number or structure
What is a single nucleotide polymorphism?
When a single nucleotide is substituted with a different nucleotide
What is a transition (type of SNP)?
A point mutation that changes a purine nucleotide to another purine (AG) or a pyramidine nucleotide to another pyrimidine (CT)
2/3 SNPs are transitions
What are the purine nucleotides?
Adenine
Guanine
What are the pyrimidine nucleotides?
Cytosine
Thymine
What is a transversion (SNP)?
Point mutation that changes a pyrimidine nucleotide to a purine one or vice versa
What is a silent single nucleotide polymorphism?
A point mutation that produces a different codon that codes for the same amino acid. This is possible because genetic code is degenerate so more than one codon codes for the same amino acid
What is a missense mutation?
A point mutation which results in a codon that codes for a different amino acid (nonsynonymous mutation)
What is a nonsense mutation?
A mutation in which a sense codon is changed to a stop codon
What type of mutation causes sickle-cell anaemia?
A non-synonymous missense mutation (SNP)
Glutamic acid substituted by valine at position 6
What is sickle cell anaemia?
Red cells lose their shape in low oxygen partial pressure and become sickle-shaped. However, there is an advantage of carrying this allele - a resistance to malaria - therefore the mutation remains widespread
What is the huge problem with insertions and deletions?
These can cause frameshift, where the addition or removal of bases changes the reading frame. Every codon following the mutation is read differently, greatly altering the resulting polypeptide
What type of indel (deletion) is less severe?
Indels of 3 rather than 1 or 3 as one amino acid is lost rather than frameshift being caused
What is a truncated protein?
A shortened non-functional protein where a mutation has caused a stop codon to appear instead of one of the codons which codes for an amino acid
What is polyploidy and what is its benefit?
Duplications in the number of chromosomes - drives evolution and speciation because genes in two sets of chromosomes can assume different functions
Causes of mutations
DNA replication mistakes Recombination mistakes Meiosis mistakes Transposons Mutagens: either chemicals or radiation
How are DNA replication mistakes usually corrected?
By editing/proofreading by DNA polymerase in a 3’ —> 5’ direction
What is the difference between mutations in germ line and somatic cells?
Germ line - individuals have mutation in all cells
Somatic cells - can give rise to cancer
What is the Ames test?
An in vitro test to detect environmental mutagens. The mutagenic potential of chemical compounds can be tested
Example: his mutant of Salmonella typhimurium - unable to grow unless histidine is present
Expose his mutant to mutagen and observe the frequency of reversion (where it can produce histamine on its own) - this tells you the rate of mutation due to the mutagen
Why is ionising radiation damaging to cells?
It produces a reactive ion species by interacting with biological molecules. Radiation can cause single or double strand breaks in DNA
UV radiation creates thymine diners (pairings) in DNA
Process of polymerase chain reaction (PCR)
- Isolate the DNA from an organism
- Heat the DNA to 95 degrees Celsius for one minute to separate the double strands
- Cool to around 60 degrees anneal short ‘primers’ to the DNA (20bp long). Primers are complementary to the start and end of the DNA being amplified
- Heat to 72 degrees, the optimum temperature for DNA polymerase
Ingredients for PCR
DNA polymerase
Free nucleotides
Primers
Template DNA
What is the benefit of using the primers that are complementary to certain DNA sequences?
They are gene-specific, so allow the isolation of a single gene
What bacteria is most commonly used for manipulation of genes in vitro?
Escherichia coli
What is a cloning vector?
An independently replicating piece of DNA into which a foreign gene can be inserted, such as a plasmid or viral genome
The vector confers the selectable phenotype onto the host cell or organism
Plasmids can be taken up by bacterial cells by the process of transformation
What is a multiple cloning site?
An area on a plasmid where there are many DNA sequences and the plasmid can be cut using restriction enzymes
What are restriction enzymes?
They recognise specific short sequences of DNA and cleave the DNA at or near that site
The recognition sites are usually 4-8 base pairs long and are palindromic
How do bacterial cells protect their own DNA from being cut with restriction enzymes?
They are protected by the addition of methyl groups to the A’s or Cs within the sequence recognised by the enzyme
Process of inserting gene into plasmid
- Restriction enzyme cuts the sugar-phosphate backbones at the recognition site. The fragment from a different DNA molecule is cut by the same restriction enzyme
- Base parking of sticky ends produces various combinations
- DNA ligase seals the strands, meaning there is a recombinant DNA molecule in the plasmid
Process of gel electrophoresis
- DNA for analysis is pipetted into small holes in the gel (an aqueous polymer such as agarose) called wells
- DNA migrates through the gel in response to an electrical field
- The pores in the gel act as a sieve, so smaller DNA molecules travel faster and further
- DNA in the gel is stained with DNA intercalating dye, such as ethidium bromide. It can slide into the twists of the DNA double helix, and is fluorescent under UV light
Measuring mRNA 1: The Northern Blot
- RNA isolated from the organism is separated on a gel
- The RNA within the gel is blotted onto a membrane that binds RNA
- A shirt piece of single stranded DNA complementary to the RNA is incubated with the membrane
- The DNA probe only anneals to the mRNA of interest on the membrane. The DNA probe is detectable
- The more abundant the mRNA, the more probe sticks to it
- The amount of probe is measured using imaging instruments
- The band thickness tells you the expression level
Measuring RNA 2: RT-PCR
The same as PCR with one additional step - the mRNA is converted to cDNA using reverse transcriptase, which undergoes PCR using primers specific to the gene of interest
A DNA-binding dye is mixed with the RT-PCR reaction, and the amount of DNA accumulating can be measured by a laser
What are microarrays?
Allow study of whole transcriptomes
Small glass/plastic slide onto which is spotted a grid of tens of thousands of unique short DNA sequences complementary to the genes of the organism
Array is incubated with mRNA or cDNA of sample
Fluorescent labelling allows detection of the amount of nucleic acid bound to each short DNA sequence on the array
Measuring amount of an individual protein: Western Blotting
- Mix of proteins extracted from an organism is separated using polyacrylamide gel electrophoresis (PAGE)
- PAGE gels are very thin and normally arranged vertically
- The protein extract is treated with detergent to give it a negative charge
- Smaller proteins travel faster and further
- The protein is transferred onto a nitrocellulose membrane
- The antibody and fluorescent label that detects antibodies are applied to the label
- Antibody attaches to protein of interest
- Darker bands indicate that more antibody has attached, so more of the protein of interest is present
