Nucleic acids- Human genome, DNA structure and replication and cancer Flashcards
1
Q
How many nucleotides in a human genome?
A
3 billion nucleotides distributed between 22 autosomes, 2 sex chromosomes (X and Y) and a small amount of DNA in mitochondria.
2
Q
Megakaryocytes
A
The parent cells of blood platelets copy DNA several times without dividing (4n,8n,16n…)
3
Q
Barr body
A
Women don’t have 46 functional chromosomes in each 2n cell.
They inactivate one copy of their X chromosome in each cell of their body and push it to the edge of the nucleus.
4
Q
Synteny
A
Where long DNA sequences (e.g. genes) are present in the same order across species
5
Q
Xist
A
X inactivation specific transcript
Xist is a regulatory RNA that switches off a copy of the X chromosome in XX cells.
6
Q
Pseudogenes
A
These are stretches of DNA that have sequence in common with functional human genes but which (look as if they) are non-functional
7
Q
Two ways pseudogenes can be generated
A
- Gene duplication- Sometimes genes are duplicated and acquire mutations that make them inactive- this is one way in which pseudogenes may arise.
- Reverse transcription can generate a different type of pseudogene.
8
Q
VNTRs and uses of them?
A
These are repeated short sequences called ‘variable number tandem repeats’.
Used for commercial, paternity and forensic testing.
9
Q
SNPs- what is a polymorphism?
A
Single Nucleotide Polymorphisms.
Multiple forms of a single gene that exists in an individual or among a group of individuals. A polymorphism is a difference present in 1% or more of the population.
10
Q
Polygenic disease
A
Whose risk increases through the many different genes acting either singly or in combination
11
Q
GWAS (Genome Wide Association Studies)
A
There may be evidence that identical twins (100% genome in common) show a greater concordance in disease risk than non-identical twins (50% of genome in common).
Some SNPs might actually cause a change in the development/disease risk of the individual.
Other SNPs may be silent, but in close genetic linkage to a site that does cause a phenotypic change.
12
Q
Cytosol
A
The thick liquid or gel surrounding the organelles
13
Q
Cytoplasm
A
The cytosol and the organelles but not the nucleus
14
Q
Polysome
A
The mRNA molecules in the cytosol become covered in ribosomes
15
Q
How many genes in the human genome?
A
20,000 protein coding genes
16
Q
Transcription
A
Process by which DNA is converted into the mRNA
17
Q
Differences between DNA and RNA
A
DNA- deoxyribose sugar RNA- ribose sugar
RNA contains uracil instead of thymine.
RNA in a healthy cell is characteristically single-stranded.
RNA differs from DNA in the structure of the sugar part of the nucleoside.
RNA is much less stable than DNA.
18
Q
3 mammalian RNA polymerases
A
Polymerase 1 transcribes ribosomal RNA.
Transcription of mRNA by RNA polymerase 2 which transcribes mRNA, microRNAs and a variety of mysterious non-coding RNAs.
Polymerase 3 transcribes tRNA.
19
Q
How is transcription started?
A
There are different promoter elements in eukaryotes including the TATA box.
This is a short run of T and A bases that can vary slightly from gene to gene.
20
Q
TATA box
A
Region of the promoter at which transcription factors bind to initiate transcription by binding the RNA polymerase to the region of the gene where transcription begins. There is more than one type of RNA polymerase in eukaryotic cells. Polymerase II is involved in the transcription of mRNA molecules.
21
Q
CpG island-
A
Stretches of DNA where there are multiple points at which C is followed by G
22
Q
Polycistronic
A
An mRNA encoding more than one protein so that the ribosome reinitiates on the same mRNA that encoded protein 1 to make protein 2, protein 3 etc.
A key difference between transcription in prokaryotes and eukaryotes is that prokaryotes produce polycistronic mRNAs.
23
Q
Polyadenylation
A
Addition of a poly(A) tail to a messenger RNA. Then mRNA is cut near the polyA sequence and a protein is made.
24
Q
What are on the ends of mRNA?
A
The 5’ end has a cap and the 3’ end a poly A tail.
25
What does the 5' cap do?
5’ cap protects the mRNA from degradation and assists in ribosome binding during translation.
26
What happens to eukaryotic mRNA?
Eukaryotic mRNA is (a) spliced, (b) polyadenylated and (c) has a 5’ cap added to it.
27
What does it mean when mRNA is spliced?
Spliced: the initial transcript has insertions in the coding sequence that arise from the transcription of introns. These sequences are cut out and the mRNA ends re-joined to make a mature mRNA.
28
What does it mean when mRNA is polyadenylated?
The initial transcript includes some sequence that is downstream (3’) of the sequence of the mature mRNA. This extra sequence is cut off and replaced with a run of ‘A’ residues.
29
What does it mean when mRNA has a 5' cap?
Guanosine ppp is added to the 5’ end of the initial transcript to make a 5’-5’ linkage that stabilises the mRNA
30
Spliceosome
Introns are removed by a complex of small nuclear RNA with proteins.
31
Alternative splicing
Alternative splicing can generate related proteins from a single gene
32
Which family of enzymes adds amino acids onto the 3′-end of newly synthesised tRNA molecules?
Aminoacyl-tRNA synthetases
33
What is a signal sequence and how is it made?
Stretch of 20 hydrophobic amino acids is called a ‘signal sequence’.
Membrane bound ribosomes start off as cytosolic ribosomes but they attach to an mRNA that happens to encode a secretory protein.
They translate the mRNA and the first bit of the protein which is produced in a stretch of hydrophobic amino acids.
Signal recognition particle binds to this and causes the ribosome to dock to the endoplasmic reticulum.
From this point onwards the newly synthesised protein is fed through a channel into the ER as it is translated.
Usually the amino terminal signal sequence is cut off.
34
How receptors are made
For receptors (a transmembrane protein) for example, it is a membrane protein that won’t be secreted. For transmembrane proteins translation/translocation continues until a stretch of approx 20 consecutive hydrophobic amino acids (red) form a transmembrane domain. This stretch leaves the channel and embeds in the membrane.
35
In Sanger DNA sequencing how is the growing chain of DNA terminated?
When a dideoxynucleotide is inserted; as it has no free 3′-OH group, the polymerase cannot continue adding bases.
36
What is needed in each reaction mix for Sanger sequencing?
The polymerase enzyme, a gene specific primer, all four dNTPs, one specific ddNTP
37
What is a DNA microarray?
An array of thousands of different DNA sequences laid out in a grid on a microchip (glass slide)
38
Give two examples of what microarrays could be used to investigate.
Occurrence of different SNPs in an individual
| Differences in gene expression between eg different samples, tumour vs normal tissue, etc.
39
How does mature mRNA reach the cytoplasm?
Through nuclear pores
40
Which amino acid is always the first in a newly synthesized polypeptide chain?
Methionine
41
To which end of the tRNA molecule is the amino acid attached?
3′end
42
What is the name of the triplet base sequence in a tRNA that binds to the codon on the mRNA?
Anti-codon
43
What are the two sub-units of eukaryotic ribosomes?
60S and 40S (Large and small)
44
What is the role of the E, P and A sites in the ribosome?
The newly arrived tRNA docks in the A site, the growing polypeptide is attached to the tRNA in the P-site. After the next polymerization reaction, the uncharged tRNA moves to the E-site and exits the ribosome.
45
What are the three stop codons that signal the termination of translation?
UAA, UAG and UGA
46
In which organelle does most post translational modification of secreted proteins take place?
Golgi complex
47
Do some regulatory RNA molecules code for proteins?
No
48
Early evidence of DNA- Griffith experiment
colonies & was harmless (non virulent), the other as ‘smooth’ colonies and was deadly (virulent).
When the heat killed smooth strain and the live rough strain were mixed, mouse died due to DNA exchange, exchanging of plasmids only DNA from the heat killed strain could produce virulence in the rough
Shows bacteria are capable of transferring genetic information.
49
Early evidence of DNA- Oswald Avery
Something in the heat killed deadly strain could transform the rough strain into the deadly strain.
Oswald Avery showed that DNA was the transforming agent since:
Out of purified DNA, RNA, protein, lipid and carbohydrate only DNA from heat killed virulent strain could induce virulence in the non-virulent strain.
50
Early evidence of DNA- Hershey & Chase
Role of DNA as the genetic material was confirmed in experiment where protein and DNA components of bacteriophage were labelled with different radioactive molecules.
Process:
• Bacteriophage are made up of proteins and DNA.
• Attach to bacteria and inject the DNA into the bacteria where replicates and uses the cells enzymes to produce new proteins to package the replicated DNA
• Forms new phages that are released when the bacteria die
- In this experiment, one bacteriophage’s protein was labelled with radioactive sulfur
- Another bacteriophage’s DNA was labelled with radioactive phosphorus
- Only the bacteria injected with the phosphorus DNA bacteriophage contained the radioactive phosphorus
- No sulfur was detected in the other group of bacteria
- Therefore DNA must contain the genetic information
51
Structure of DNA
```
Erwin Chargaff (1952) - studied base composition in DNA in different species
Found ratio of G:C and A:T was always 1:1 suggested that these bases were paired
```
Watson and Crick (1953)/ Rosalind Franklin showing the X-ray crystallography- x-ray diffraction
Electron microscopy showing the double helix and anti-parallel.
DNA structure:
* Antiparallel strands form double helix
* Sugar phosphate backbone
* Base pairs join complementary strands together by hydrogen bonding (adenine with thymine, guanine with cytosine)
* Unit is the nucleotide (base+sugar+ phosphate)
* In DNA sugar is deoxyribose
* Backbone of DNA is sugar phosphate polymer
* Adjacent deoxyribose sugars are linked by phosphodiester bonds
* 5’ and 3’ ends give DNA strand directionality
* 3 HB between G and C 2 HB between A and T
* DNA is tightly coiled with help of proteins to fit its considerable length into the nucleus
* DNA plus (histone) proteins known as chromatin
* Active genes are more loosely coiled than silent ones
52
DNA replication
DNA replication is semiconservative. Each new double stranded molecule contains an original (template) strand and a newly synthesised complementary strand.
53
What is the replication fork?
At the replication fork:
– helicases unwind the double stranded DNA to allow replication to occur
– Single strand binding proteins stabilise the denatured DNA
– DNA primase synthesises a short RNA primer to allow replication to commence
– DNA polymerase carries out the elongation of the new strand of DNA, which forms by complementary base pairing to the template strand
54
In which direction are nucleotides added to the newly forming DNA strand? This is called the direction of replication.
5′ to 3′. The 5′ end has a free phosphate and the 3′ end a free hydroxyl group. The free 3′ hydroxyl group is required for DNA polymerase to add on the next base, which is the reason for the direction of DNA synthesis.
55
In which direction is the template strand orientated with respect to the growing strand during DNA replication?
3' to 5'
56
What enzyme catalyses the formation of a new phosphodiester bond?
DNA polymerase
57
What is the source of the energy that drives DNA replication?
The substrates for new nucleotides are the deoxyribonucleoside triphosphates (dNTPs). As the new nucleotides are added to the growing DNA chain, pyrophosphate is released, with the release of energy that is used to form the phosphodiester bond between nucleotides.
DNA synthesis requires a pool of all four deoxy nucleoside tri-phosphates (dNTPs).
58
Leading and lagging strands
Because DNA can only be synthesised in one direction, it must be made as short strands on the lagging strand, each one primed with a new RNA primer.
59
What enzyme replaces the RNA primers with DNA?
DNA polymerase I
60
What enzyme seals the gaps between fragments?
DNA ligase
61
DNA replication error rate
An error occurs about once every 100,000 nucleotides inserted into the growing strand of DNA (1 in 10^5).
62
How is DNA replication error rate reduced?
DNA polymerases have 3′ to 5′ editing function to remove incorrectly inserted bases. This reduces error frequency to around 1 in 10^7. A further check for mismatched bases by other enzymes helps to reduce the overall error frequency to 1 in 10^9 bases.
63
Germ cell
A cell containing half the number of chromosomes of a somatic cell and able to unite with one from the opposite sex to form a new individual; a gamete
64
How is DNA damaged?
DNA damage by endogenous and exogenous chemicals.
Endogenous- growing or originating from within an organism
Exogenous- growing or originating from outside an organism
65
How is DNA damage repaired?
- DNA repair proteins remove the damage before replication occurs.
- Base excision repair proteins cut out damaged bases- they are specific to specific types of damage.
- Nucleotide excision repair proteins are less specific and cut out sections of the damaged DNA strand.
66
What is spontaneous deamination of cytosine?
Removal of an amine group. This occurs slowly in aqueous solution and changes the sequence of the DNA strand.
The enzyme uracil N-glycosylase recognises uracil in DNA and cuts it out. This is a base excision repair enzyme (which is very specific for its substrate).
67
Name three antibiotics that target prokaryotic ribosomes
E.g. tetracycline, streptomycin and chloramphenicol
68
Cancer
A disease caused by an uncontrolled division of abnormal cells in a part of the body
69
Dedifferentiation
Cells regress from a specialized function to a simpler state reminiscent of stem cells
70
Characteristics of cancer cells
- Uncontrolled growth
- Loss of contact inhibition
- Immortal
71
Contact inhibition
A regulatory mechanism that functions to keep cells growing into a layer one cell thick (a monolayer).
Cancer cells divide but don’t stop dividing, they keep dividing building on top of each other. The cells lose control.
72
Hyperproliferation
An abnormally high rate of proliferation of cells by rapid division
73
Hyperproliferation
Normal cells-> hyperproliferative cells-> early adenoma-> late adenoma-> carcinoma
74
Genetic instability
A lot of mutations
75
Point mutations (or base substitutions)
Smallest change in DNA sequence that can give rise to a change in gene function. They can result in an amino acid substitution (missense mutation) or can introduce a stop codon (nonsense mutation) into the coding sequence of a gene resulting in a truncated protein product
76
Frameshift mutation
Gain or loss of one or two base pairs that results in a shift in the reading frame of a gene transcript
77
Gene amplification
A cell having anything up to a hundred copies of a gene that it would normally only have two copies of
78
Chromosomal translocations
Result in genes being moved to a more transcriptionally active region of the chromosome, or can result in two genes being recombined into a new gene fusion
79
Aneuploidy
Any departure from the normal structure or number of chromosomes
80
Mutations can do two things to genes
- They can disrupt the coding sequence sufficiently to stop the protein product from functioning normally
- They can actually make the protein more active by improving the amino acid sequence, or by allowing more copies of the protein to be made.
Can lead to activation (gain of function) or inactivation (loss of function) of gene product
81
Mutation induction requirements
- Chemical modification of DNA and/or
- Replication of DNA
- Resulting in misincorporation by DNA polymerase
82
Causes of cancer
- DNA polymerases make mistakes. Results in the accumulation of genetic variation or polymorphisms in coding and non-coding sequences in the genome. Some of these changes are deleterious and are known as mutations.
- The presence of chemical modifications (miscoding or non-coding adducts or lesions) in the DNA increases the chance for polymerases to make mistakes (point mutations) by misincorporation, or can cause the polymerase to stall leaving a break in the DNA strand that can end up as a double stranded DNA break – a substrate for deletions, insertions or translocations
83
Hallmarks of cancer cells
* Invasive and metastatic- have the ability to break cell walls and spread/ go into the lymphatic system to produce a new tumour
* Independent of positive growth factors- cancer cells will divide even if they aren’t told by a positive growth factor
* Resistant to negative growth factors- ignores and carries on dividing
* Immortal
* Resistant to apoptosis- Even if have damage they carry on dividing
* Angiogenic- can promote the growth of new blood vessels to provide the nutrients so the tumour can keep growing
84
Angiogenesis
The formation of new blood vessels
85
Oncogenes
Mutated versions of normal genes that play a key role in promoting growth and division of cells. Mutations lead to increased activity/ inappropriate switching on of cell division
86
Tumour suppressor genes
Genes that play a role in controlling growth/protecting cells against damage. Mutations that inactivate both copies of these genes are involved in carcinogenesis
87
Carcinogenesis
The initiation of cancer formation
88
Cancer syndromes
Genetic disorder in which inherited genetic mutations in one or more genes predispose the affected individuals to the development of cancers
89
Environmental agents that cause cancer
-Biological:
• Hepatitis B virus (liver cancer)
• Human papilloma virus (cervical cancer)
• H. pylori (stomach cancer)
-Chemical:
• Cigarette smoke (lung, bladder, others)
• Heterocyclic amines in cooked meat (colon)
-Physical:
• UV in sunlight (skin cancer)
• X-rays/γ-rays (radiotherapy related leukaemia)
90
Genotoxic
Damage of DNA and cause mutations
91
Non-genotoxic
Induce cell division and DNA replication without being mutagenic (no gene damage)
