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Molecular Anatomy Of Genes And Genomes Flashcards

1
Q

What is a main difference seen between prokaryotic and eukaryotic genomes and genes

A

They are different sizes. Eukaryotic are bigger with more base pairs and number of genes

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2
Q

What are the parts of a eukaryotic gene what what is each part (6)

A

Promotors/ control regions- determine if a gene is on or off in a tissue and its level of expression- trnscription factors bind to this region to do this
Exon- part of a gene that is expressed after transcription/ is also translated
Intron- part of a gene that is transcribed only so not expressed. Is cut out in splicing
5’ Cap- first small sequence of base pairs which allows recognition by a ribosome
PolyA tail- for stability and recognition at the end of a gene
5’ and 3’ UTR- determine how long mRNA stays in an cell and how quick/ when translation occurs. Not translated

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3
Q

What is splicing

A

The process where introns are removed from the gene whcih is to be expressed so only exons are expressed

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4
Q

Roughly how much of the genome codes for proteins

A

~1.2%

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5
Q

How much of the human genome is repetitive DNA and what are the two types

A

48%- highly repetitive DNA sequences and transposons

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6
Q

What are non protein-coding genes

A

tRNA genes, ribosomal RNA, microRNA and long non-coding RNA with regulatory roles

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7
Q

What are highly repetitive sequences in the human genome and how much of the genome do they make up

A

Short repeats of highly repetitive DNA (short-tandem repeats or satellite DNA). Make up ~3% of the genome. The repetitive sequences vary between unrelated individuals and is therefore, the basis for DNA fingerprinting as the differences can be compared between related people

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8
Q

How do highly repetitive sequences occur

A

By replication slippage/ by chance

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9
Q

Two examples of things which are compossed of highly repetitive DNA in humans

A

Centromere- has no defined sequence and typically consists of large arrays of repetitive DNA (Satellite DNA)
Telomeres- repeat sequences added by telomerase to protect ends of chromosomes

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10
Q

What are transposons, what are the four types and how much of the genome does it make up

A

Moderately reprtitive DNA. LINEs, SINEs, LTR retroptransposons and DNA transposons. Make up ~45% of the human genome

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11
Q

How much of the genome is transcribed and what are the parts that do not encode proteins

A

~60%. Non-coding RNA are tRNA, ribosomal RNA, microRNA and long non-coding RNA. These make up most of the transcribed genome

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12
Q

How many base pairs does the human genome consist of

A

~ 3,000 million

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13
Q

What is the purpose of packaging DNA into chromosomes

A

Compacts DNA
Protects DNA from damage
Ensures replicaed DNA is properly segregated
Provides overall organisation which facilitates recombination and gene expression

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14
Q

What is the first step to DNA packaging

A

DNA is wrapped around positively charged histone proteins to make nucleosomes

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15
Q

What is the composition of a nucleosome and its look when a picture is taken

A

Core of 8 histone proteins
DNA is wrapped 1.65 times around the outside
Linker DNA links nucleosomes as it connects to the DNA wrapped aorund histone cores
Looks like beads on a string

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16
Q

What us histone H1

A

They attach to nucleosome DNA and indice tighter wrapping around the nucleosome to compact the nucleosome DNA into 30n, chromatin fibres

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17
Q

What are chromatins/ loops of DNA connnected to and what does it contain

A

Attached to a protein scaffold which contains topoisomerase II and specific proteins such as condensin

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18
Q

How does the cell organise DNA so that the stuff that is needed for replication is accessible and the stuff not needed to protected from damage

A

Histone tails- chemically modified by enzymes which determine if DNA is tightly compacted/ turned off (heterochromatin) or if the genes are loosely packed and found around RNA polymerase for transcription/ turned on (euchromatin).
Histone tails can be modified to allow for switching between the two states

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19
Q

What are the seven types of enzymes involved in DNA replication

A

DNA topoisomerase
DNA helicase
Proteins that prevent reannealing of DNA (single strand binding)
Enzymes to synthesise RNA primers (DnaG primase)
DNA polymerase
Enzymes to remove RNA primers (polymerase I exonuclease 5’ to 3’ in prokaryotic, RNAse in eukaryotic)
Ligase to covalently link okazaki fragments

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20
Q

What are three substrate requirements for DNA polymerase

A

Template strand must be there to be copied
Primer- DNA polymerase I cant initiate DNA synthesis and can only extend a preexisting chain
3’ hydroxyl end free for addition of nucelotides

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21
Q

What shape can DNA polymerase I be compared to and its important parts

A

A hand. Palms and fingers

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22
Q

What does the ‘palm’ of the hand of DNA polymerase I do

A

Is a catalytic site which provides the correct environment for the reaction. Specific amino acid side chains and two magnesium ions

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23
Q

Why can only correctly base paired dNTPs be added during DNA replication

A

Incorrect base pairing causes inspecificity. If there is correct base pairing, the OH is close enough to the phosphodiester bond to add the phosphate on

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24
Q

How does distinguishing between rNTPs and dNTPs occur

A

There is a discriminatory amino acids found in DNA polymerase I which detect the extra OH from rNTPs which will distort the DNA if it binds meaning the phosphate will be too far away from the other nucleotides and cant pair

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25
What do the fingers of DNA polymerase I do
Bind to incoming dNTP and fold over if the base pair is correct. They also twist the template so that only one base is in the active site at a time
26
How are DNA polymerase I mistakes corrected (brief overview)
Polymerase I has exonuclease activity which removes incorrect nucleotides and allows for correct ones to bind.
27
If there was no exonuclease, how many mistakes would be made in DNA replication and how many mistakes are made with exonuclease
DNA polymerase incorporates a mistake every 100,000 bp | With exonuclease, there is a mistake every 10,000,000,000 bp
28
What does it mean when it is said that DNA replication is semidiscontinuous
Lagging strand requires RNA primers via primase. Primase synthesises ~11 nucleotide RNA in vivo and associates and dissociates every time a primer is synthesised. The DNA can then be added to the lagging strand via okazaki fragments
29
How are primers removed and how is the missing areas fixed
Removed by polymerase I 5’-3’ exonuclease and the missing regions are sealed by DNA ligase
30
How many subunits does helicase have
6
31
How does DNA helicase work and what is required for it to work
Unwinds at the replication fork, must be loaded onto DNA and are processive. Require ATP to work
32
What are single strand binding proteins and what do they do
Proteins that prevent strands from reannealing/ coming back together
33
What do topoisomerases do
Remove supercoils in DNA during DNA replication and can add coils in DNA for condensing
34
What is the s.iding clamp in DNA replication
Binds to DNA polymerase III to prevent dissociation from the DNA strands
35
What is a replisome
Multi-protein complex which includes two DNA polymerase III enzymes and sliding clamp (aswell as clamp loader)
36
What must happen to the lagging strand for the replisome to move in the 5’ to 3’ direction
The lagging strand must loop around so that the replisome can work in the correct direction as the lagging strand is in the 3’ to 5’ direction which will not work
37
How does DNA replication start and end in prokaryotes
Begins at the origin of replication and finishes at specific, defined points
38
What are the reasons why DNA replication occurs with little mistakes (4)
Cells remain constant levels of dNTPs (dATP, dCTP, dGTP, dTTP) Polymerase reaction has high fidelity due to it occurring in two stages- incoming dNTP base pairs with template while enzyme is open and polymerisation occurs once ‘fingers’ are closed around positioned dNTP Proof reading ability of DNA polymerase DNA repair systems in the cell that identify errors before DNA is replicated again
39
What are the several DNA polymerases sued and what does each one do
Alpha- initiates DNA synthesis Delta- synthesis of the lagging strand (requires sliding clamp to come on and off) Epsilon- synthesis of the leading strand
40
What is a amjor difference between prokaryotic and eukaryotic DNA replication origins
Prokaryotic is originated form one site, eularyotic has many origins of replication
41
What are telomeres
Sequences of repetitive DNA at the ends of chromosomes to protect the ends of DNA from degradation because of the gap that is not filled on a replicated chromosome after replicatiom
42
What is telomerase
Enzyme that operates at the end of chromosomes to extend their length/ makes telomeres. Consists of a protein and an RNA sequence which serves as a template for synthesising telomere DNA. Maintains telomeres at the ends of the DNA thread making it possible to copy the entire chromsome to its very end each time the cell divides
43
What would happen to chromosomes if telomerase was not present
The chromosome is shortened each time a cell divides until the telomere DNA is erroded and the chromosome is damaged
44
What happens to the telomerase enzyme when someone has cancer
The activity of telomerase is increased allowing for more rapid cell division
45
Which end of a DNA strand does telomerase extend
The 3’ end
46
What are the steps in PCR
Denature DNA/ separate strands (by denaturing H bonds between nucleotides) at 95°C Add primers and anneal at 60°C Extend chains with Taq Pol and dNTPs at 72°C Repeat
47
What are the two types of primers used in PCR
Forward and reverse
48
What is a dideoxynucleotide and what does it do
Eg ddATP, ddGTP, ddCTP, ddTTP They remove and OH group from their particular nucleotide which prevents another from attaching, therefore, synthesis of a DNA strand stops where that specific nucleotide is and you get different fragments in different sizes
49
What does dye label do with dideoxynucleotide sequencing
shows the specific nucleotides based on their colour on the agarose gel. Detected under a laser beam
50
Limitations to dideoxynucleotide sequencing
Sequences can only be determined in approximately 400-800 bp chunks (reads)= there is an upper size limit Cant sequence something forever/ cant sequence an entire genome, need to do short fragments at a time
51
How does next generation sequencing work
Prepare DNA library- ligate DNA adapters (known sequences) to the ends of each DNA fragment for PCR amplification Capture a single DNA molecule into one micro-well. There are billions of wells so billions of molecules can be sequenced at once Undergo PCR for each DNA molecule 1-hundreds to get enough signal to detect Sequence
52
How does the actual sequencing in next generation sequencing work
Add dGTP and polymerase. A reversible blocking group is added so that other nucleotides cant attach and dye is also added. Goes through laser imaging to see the colour and therefore, the base detected Add chemical to remove 5’OH block and dye Add another dideoxynucleotide eg dTTP and go through same process. Another colour will be detected
53
How does RNA sequencing (RNAseq) work
``` Isolate RNA RNA to cDNA Frgment cDNA and made library (by putting known sequences on the ends of unknown fragments and making primers for these known sequences) Sequence Map sequence reads ```
54
How is the map of sequence reads used
Exonic read overlaps can be used to form the correct DNA sequence we are looking for. Also, the prevalence of certain exonic reads (as there tends to be overlaps) tells us how much that particular sequence/ gene is expressed
55
How to made cDNA from reverse transcription
Add reverse transcriptase (viral DNA polymerase that uses RNA as template, primer and dNTPs) Remove RNA template Copy new DNA strand
56
What is real-time PCR
Computer based analysis of the cycle-flouresence time course. Detects the amount of flourescence from each PCR cycle. Flouresence comes from a dye that gets inbetween two DNA strands. The higher the flourescence, the more dsDNA that has been made
57
What does Ct mean and its relstion to real time PCR
The number of cycles required for the fluorescent signal to cross the threshold and be detected by real-time PCR. Ct values are inversely proportional to the amount of target nucleic acid in the sample… lower the Ct value, the more that gene is expressed
58
Why does a real-time PCR curve flatten off
Because PCR continues until there are no more primers or nucleotides left. So once all these are used PCR does not continue and the real-time curve flattens off
59
Why is a reference gene used alongside the testing gene in real-time PCR
A reference gene is expressed at a similar/ same level in all tissues and at all conditions. Every cell needs the same amount Acts as a control as it shows that there is the same amount of teh wildtype and mutant genes being used if there is the same amount of the reference gene in each one
60
If there is similarity in gene sequence, what does that mean for the function between organisms
Sequence similarity ≈ function similarity | Comparative sequence analysis
61
What are common research questions when searching sequence databases
To find a particular gene in a particular sequence To find out a possible function of a gene To find genetic variation in a gene of interest
62
How is DNA made into clones in molecular biology
Recombinant DNA technology- mRNA inserted into vectors to be put into bacterial cells (mRNA as bacteria dont have introns so mRNA only has exons)
63
What does transcriptome mean
All of the transcripts produced by a cell, tissue or organism

BIOC221 (5 decks)

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