Flashcards in Autumn Term Deck (120)
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1
What are the stages of meiosis?
Prophase
Prometaphase
Metaphase
Anaphase
Telophase
2
What are the stages of interphase?
G1, S, G2
G1 > G2
3
What is G0?
A dormant state
Most of the cells leave the cycle to be in this phase
Allows for the terminal specialisation of cells
4
What are the major checkpoints?
G1S: checks the size of the cell, checks whether DNA is damaged
G2M: checks that there is double the DNA, checks whether DNA is damaged
M: checking that the chromosomes have engaged with the spindle fibres
5
What is a nucleosome?
Two copies of four histone proteins with DNA coiled around it
6
What is the structure of a chromosome?
Two chromatids are separated by a centromere
Once separated, they form two chromosomes
One of these chromosomes is duplicated and it forms two pairs of sister chromatids
7
What is a karyotype?
The number, size and shape of chromosomes in an organism
8
What is the process of interphase?
Chromosomes uncoil, forming chromatin
Cell growth
9
What is the process of prophase?
Chromosomes coil and condense
Centrioles divide and move apart
10
What is the general process of prometaphase?
Chromosomes are double structures
Centrioles reach the opposite poles
Spindle fibres are formed
Chromosomes have not engaged yet
11
What is the process of metaphase?
Centromeres align on the metaphase plate (equatorial line)
12
What is the process of anaphase?
Centromeres split and daughter chromosomes migrate to opposite poles
13
What is the kinetochore?
It is responsible for the movement of sister chromatids
Protein complex
14
What is the process of telophase?
Daughter chromosomes arrive at the poles
Cytokinesis commences
15
What is the process of plant cytokinesis?
Formation of two square membranes
In between is the middle lamella
16
What is the process of animal cytokinesis?
Forms a strangulation ring that separates two daughter cells
17
What is a tetrad and dyad?
Metaphase forms two tetrads
Division creates two dyads
Meiosis II creates monads
18
What are the stages of prophase?
Leptonema
Zygonema
Pachynema
Diplonema
Diakinesis
19
What is leptonema?
The start of prophase
Chromosomes begin to condense
Homologous pairs form
Homologous search
20
What is zygonema?
Synaptonemal complex
Brings homologous pairs into close proximity
Pairs of homologous chromosomes
Causes their segregation
Homologues are synapsing
Non-sister chromatids remain connected
Key event = synaptonemal complex
21
What is pachynema?
Crossing over between non-sister chromatids
Key event = synapsis
22
What is diplonema?
A chiasma is where non-sister chromatids are joined
Key event = chiasma, start to pull apart
23
What is diakinesis?
Centromeres are replicated
Condensed chromosomes
Key event = centromere attachment
24
How many chromosomes are there?
23 including the sex chromosome
25
What are chiasmata?
Where DNA is transferred during crossing over
26
Do sister chromatids have the same alleles?
Yes
27
What is a testcross?
A way to determine whether an individual displaying the dominant phenotype is homozygous or heterozygous for that trait
The parent must be heterozygous recessive for all three genes under consideration
28
What was Mendel's hybrid cross?
9:3:3:1
29
Human pedigree charts
Circles = female
Squares = male
Assume that all individuals marrying into the family are normal homozygous
30
Which parent passes on the x-linked mutation?
Fathers always give their y to their sons so the mother will always give the x linked mutation
31
How do you calculate genetic risk?
Work out the probability of both parents having the allele then multiply down the line
32
What is a plasmid?
A small circular piece of DNA that is separate from the chromosome and can replicate independently
33
What is a recombinant gamete?
A gamete which contains a different combination of alleles compared to the parental ones
34
What is the percentage recombinance in a single crossover?
Maximum 50% because only two chromatids swap
The other chromatid stays the same
35
What is one map unit?
One map unit
= 1% recombination between genes on the same chromosome
= 1 centiMorgan
36
NCO
Non-crossover chromosomes
37
What are the most abundant classes in a testcross?
The parental classes
38
How do you determine the middle gene?
Focus on the least abundant class (DCO)
Swapping the gene in the middle from the parental chromosomes produces the two smallest classes
39
How do you determine the map distances?
The distance between alleles
Calculated by selecting all recombinants between these two genes
When the two alleles are next to each other, add those numbers to the DCO numbers
Then divide by the total number of offspring then multiply by 100
If a table doesn't show a group, write them in as 0
40
How do you work out the chance of recombination from the centiMorgans?
10cM difference = 10% chance
41
How do you work out the percentage of gametes expected to be a double crossover?
Multiply the two distances between genes together
The distance between two genes includes the probability of single and double crossovers so you need to minus the double crossover percentage from it to find the single crossover percentage
42
What is transcription performed by?
RNA polymerases (RNAPs)
RNAPs use DNA as a template and ribonucleotide building blocks
RNAPs move along the template strand in the 3' to 5' direction
The RNA transcript is synthesized in the 5' to 3' direction with nucleotides added to the 3' end
The 5' end is the phosphate end, the 3' end is the hydroxyl of the pentose sugar
43
What is a transcription bubble?
RNA opens up the double helix to form a transcription bubble
44
What are the two strands in a double helix?
Template and non-template
The template strand is called the coding strand because it will have the same sequence as the RNA produced from the template strand
45
What is the process of translation?
Takes place on the ribosomes
Ribosomes move along mRNAs in the 5' to 3' direction
Genetic code is read in a triplet codon
tRNAs bring amino acids to the ribosome
Anticodons on tRNAs pair with codons on mRNA
Ribosomes catalyze peptide bond formation between amino acids
46
What should the open reading frame contain?
A series of triplet codons coding for amino acids
It should start with a start codon and end with an end codon
47
What is degenerate code?
Where more than one triplet code codes for the same amino acid
48
What is an anticodon loop?
Base pairing with the codon
Antiparallel configuration with the 5' codon end pairing with the 3' end of the anticodon
49
What is the structure of ribosomes?
Composed of protein and RNA
Three important sites: E, P, A
A site: decoding centre, where incoming tRNA would pair with its codon, this sets up the amino acids perfectly for peptide bond formation
When a peptide bond forms, the join between the amino acid and a new tRNA molecule is broken and the chain is transferred onto the A site amino acid
50
What are the points of gene control expression?
Before transcription: which genes are transcribed
Controlled at the point of RNA stability: unstable RNA breaks down quicker so can be used for shorter time periods
Protein stability: if the RNA is unstable, the protein will be allows for a high turnover
51
What is the key regulation of transcription?
Regulating the interaction of RNA polymerase with the promoter region
Regulatory proteins (transcription factors) play a key role
52
What is the positive regulation of transcription?
Activating transcription factor proteins are required to turn transcription on
All promoter sequences are not the same
Promoters can be weak or strong in attracting RNA polymerase
Naturally RNA polymerase doesn't have a strong affinity for the promoter sequence
In the presence of an activating transcription factor, RNA polymerase binds to the promoter
53
What is the negative regulation of transcription?
A gene is expressed unless a repressor protein is present to turn transcription off
In the presence of a repressor, the access to the promoter is blocked
54
What is the prokaryotic control of gene expression?
Stimulus - Regulatory protein - target gene(s)
Regulatory genes come in two forms: one is able to bind to DNA and regulate gene expression and the other form can not
The promoter is where positively acting transcription factors bind
Promoter - Operator - ORF
55
What is the operator?
A DNA sequence where the repressor protein will bind which usually overlaps with the transcriptional start site
So putting a repressor here would block all transcription
56
Operons
Where there is a single regulatory region and multiple linked genes
The multiple linked genes will be transcribed as a single mRNA called polycistronic mRNA
Several proteins will be produced from this sequence because the ribosome will translate them separately because each gene will have a start and stop codon
57
Lac operon
Allows the synthesis of lactose metabolising enzymes in E.coli when lactose is present as a carbon source
The enzyme responsible for metabolising lactose is B-galactosidase (cleaves lactose into glucose and galactose)
Structure = promoter, operator, lacZ, lacY, lacA
58
How is negative regulation achieved via the lacI repressor protein?
In the absence of lactose, lacI binds to the operator and prevents transcription of the lac operon
The lacI repressor is able to bind to lactose
This leads to a change in the shape of the lacI repressor and it can no longer bind to DNA
This type of regulation is allosteric
59
Lac I defective mutation
Mutation within the lacI gene
Nucleotide sequence changes in the gene prevents the protein from acting as a repressor
The mutation gives rise to constitutive expression of the lacI operon which is expressed even in the absence of lactose
60
Operator constitutive (Oc)
Mutation in the operator sequence
The lacI repressor can no longer bind to the operator
61
Why is glucose a better carbon source than lactose?
If glucose is present, the lac genes are not expressed even if lactose is present
Controlled by the CAP transcription factor
E.coli uses glucose first them enters a lag phase of no growth then uses lactose as a carbon source
During the lag phase, lac operon is being switched on so that lactose can be used
62
Inverse relationship between glucose levels and cAMP levels
Cyclic adenosine monophosphate = cAMP
A high glucose concentration inactivates adenylate cyclase (an enzyme that catalyzes the formation of cAMP from ATP)
Once glucose becomes more used up, cAMP is formed more
63
The cAMP CAP complex activates transcription
CAP is a catabolite activator protein: a positive regulator of transcription
Allosteric regulation
The complex binds to the promoter region
When glucose is used up, cAMP levels will rise and so the CAP transcription factor will be active
The lac operon will be transcribed
Initially levels of cAMP will be low because glucose levels are so high
Even though lactose is present, the lac operon won't be transcribed because the CAP transcription factor won't be activated
64
What is RNAP?
RNA polymerase
65
Difference between prokaryotes and eukaryotes in transcription and translation
Prokaryotes undergo coupled transcription and translation
Eukaryotes undergo spatial separation
66
What are polysomes?
Multiple ribosomes translating a single mRNA
67
What are the problems with RNA polymerase binding?
The nucleosome structure gets in the way of RNA polymerase binding
When genes are activated, the nucleosomes are repositioned to be further apart
This allows access for transcriptional machinery
Opening up of the chromatin structure requires protein complexes that can perform chromatin remodelling
68
How is chromatin opened up?
There are regulatory proteins that are able to bind to target sequences on DNA even when it is packaged into a tight nucleosome configuration
These proteins can modify the nucleosomes or histone components to remodel the chromatin
69
Does RNAP have a high affinity for DNA?
No
Even when RNAP has access, it alone has a low affinity for promoter regions and needs additional protein transcription factors to assemble
70
Difference between eukaryotic and prokaryotic promoters
Eukaryotic ones are more complex and contain multiple binding sites for transcription factors
71
What is the core promoter?
Where RNAP and general transcription factors bind
Establishes the start site for transcription initiation
Includes the TATA box and initiation site
72
Promoter proximal enhancer elements located?
They vary from gene to gene
Occur upstream from the promoter
Important in determining where, when and at what level transcription of that gene will take place
Binding sites for regulatory proteins, some of which will be involved in opening up chromatin whereas others will be involved in recruiting RNA polymerase
73
What are distal enhancer elements?
Located at a distance from the transcriptional start site
Increases the level of transcription
Can be upstream or downstream
Can be very far away from the start site
Binding sites for transcription factor proteins
74
Do prokaryotic genes have regulatory proteins?
Most don't
75
Why can the whole sequence including the distal enhancer elements be looped round?
They can loop round and help to stabilise RNA polymerase at the promoter
This allows them to be downstream because they can loop back to affect the region
76
RNA processing
Premature RNA transcript to mature mRNA
Eukaryotic mRNAs undergo specialized processing evens at their 5' to 3' ends
Structure = 5', ORF, 3', polyA tail
mRNA has to move out of the nucleus so needs to be more stable than their prokaryotic counterparts
77
What does translation involve?
Recognising the 5' cap
78
Eukaryotic splicing
RNAP produces the primary transcript (pre-mRNA)
Pre-mRNA is processed (removing introns)
Exons are fused
Splicing removes introns from the pre-mRNA
79
Spliceosome
Splicing takes place in the nucleus
The spliceosome is a protein-RNA complex which splices pre-mRNA
Finds exon-intron boundaries
80
What is the benefit of the intron exon structure?
It increases the coding capacity of genomes without having a larger genome
81
What is alternative splicing?
Introns can be spliced in more than one way
This creates different combinations of exons in the final mRNA
This increases the number of gene products derived from the genome
Alternative protein isoforms = same gene forming different proteins
82
Missense mutation
Amino acid change
83
Silent mutation
No amino acid change
84
Frameshift mutation
Insertion or deletion of bases that is not a multiple of three, shifts the reading frame
85
Nonsense mutation
Changes a codon for an amino acid to a stop codon
86
Loss of function mutation
Usually recessive
Complete loss or reduction in protein function
87
Gain of function mutation
Usually dominant
Involves either an increase in protein function, the acquisition of a new function or acquiring an ability to interfere with the function of the wild-type protein
88
Constitutive gene
A gene that is transcribed continually
89
Facultative gene
Only transcribed when needed
90
Negative regulator
Normally repressing transcription of its target genes
91
Gfp
Green fluorescent protein
Emits green light when you shine blue light on it
92
What are the starting materials for horizontal gene transfer?
DNA containing the region of interest
Cloning vector (bacterial plasmid) containing the cloning site and the restriction enzyme recognition sites
93
Cloning vector cutting
Choose an enzyme which cuts the described sequence and the bacterial plasmid
Check that the sequence being cut doesn't also lie inside the desired sequence
Cutting the DNA using one enzyme means the DNA can be inserted either way round
For directional cloning, use two restriction enzymes
Sticky ends = breaks hydrogen bonds
94
PCR amplification of the promoter region
Polymerase chain reaction
Heat up the DNA to break the hydrogen bonds and forms ssDNA
Use a PCR primer which binds at the very start and end of the desired sequence
The desired sequence needs to have the same ends as the vector
Add some sequences to the primers that match the restriction endonucleases with some nucleotides either side
The nucleotides are added because restriction endonucleases don't like to cut at the end of the sequence
95
Ligation of vector and insert
Incubate vector and insert
There will be nicks present in the DNA where the strands aren't properly connected
If incubated with DNA ligase, the enzyme can seal those nicks
96
Transformation and selection
E.coli cells can be made temporarily competent by applying a short electrical pulse
Mix E.coli with the ligation products then electroporate the cells
Some of the cells will take up the vectors
Some cells will take up uncut or empty vectors
The majority of cells will not take up any vectors
Treat the cells with an antibiotic because the plasmid taken up will contain the gene for resistance against a specific antibiotic
Blue / white selection: use the gene that the inserted section interrupts to separate the two e.g. X-Gal and kill the blue colonies
97
Diagnostic restriction digests
Cut the cells with the original restriction endonucleases
Work out all the possible sizes of DNA segments if cut with enzymes
Each will create a DNA fragment
This will show you how pure the samples are because the fragments can only be from the desired plasmid
98
DNA sequencing
Use a sequencing primer which is close to where the insert is so the sequence synthesized is the insert
Use small amounts of ddNTPs: nucleotides that don't have the 3' OH and therefore, if they get incorporated into the strand, they stop synthesis
Different ddNTPs are labelled with different fluorescent markers
Therefore lots of strands will be labelled according to the nucleotide which was incorporated last
These strands are separated by gel electrophoresis
Reaction components: DNA template, primer, DNA polymerase, dATP, dCTP, dGTP, dTTP, small amounts of ddNTPd with fluorochromes
DNA synthesis occurs from 5' to 3'
Direction of DNA movement = 3' to 5'
Restriction endonucleases which recognise longer sequences are better because longer sequences are less likely to occur in a sequence
99
Difference between prokaryotes and eukaryotes in terms of DNA
Prokaryotes have circular chromosomes
Eukaryotes have linear, nuclear chromosomes
Prokaryotes have compact genomes, eukaryotes have very large genomes
Gene density varies along the chromosome
Prokaryotic genes lack introns and often arranged into operons
Eukaryotic genes have introns and very large intergenic regions associated with gene regulation
100
What is horizontal gene transfer?
The non-productive transfer of genetic material from one organism to another
101
What are the three types of horizontal gene transfer in bacteria?
Transformation: uptake of DNA from the environment which is incorporated into the genome
Conjugation: refers to the direct transfer of either plasmid or genomic DNA from one bacterium to another, most common type, unidirectional, F+ are strains that donate, F- are strains that receive
Transductive: transfer of genetic material between bacterial cells by bacteriophages
102
What is a cloning vectors?
DNA molecules that are used to hold a piece of DNA of interest so that it can be maintained and replicated in an organism
103
Are E.coli competent?
E.coli aren't naturally competent
104
What are restriction enzymes?
Enzymes produced by bacteria to defend against the bacteriophage infections by recognising palindromic sequences
They cleave the phosphodiester backbone
105
What is the blue/white selection?
The lacZ gene codes for beta-galactosidase which catalyses the reaction of the colourless substrate X-gal into a product that becomes oxidised into a blue product
The integration of the desired sequence will disrupt this gene so cells carrying the vector will appear white on a plate treated with X-gal
The other cells will appear blue
106
What is the process of PCR?
Denaturation: heating of DNA to 95 degrees, denatured into single strands
Hybridisation / annealing: lowering of the temperature to 55-65 degrees, allows for the annealing of DNA primers that were designed to bind to specific regions of the template DNA
Extension: the temperature is set to 72 degrees which is the temperature at which the taq polymerase synthesizes DNA by adding nucleotides to the 3' end of primers
107
What does a viral reverse transcriptase enzyme do?
It uses RNA as a template to synthesise the complementary DNA
The reverse transcriptase reaction can be followed by a normal PCR to amplify the sequence
108
How do you work out the many different gametes generated from the random arrangement of tetrads at the metaphase plate?
2 to the power of the haploid number
This is because homologous pairs line up along the equator
109
Types on inheritance
Recessive
Dominant
Autosomal
Sex-linked
110
What is the start codon?
Methionine
Met
111
Difference between codon and anticodon?
The anticodon is antiparallel to the codon
It is a reflection of the base pairs of the codon
112
What is the Shine-Dalgarno sequence?
Ribosome binding site on prokaryotic mRNA
113
Define polyA tail
A chain of around 200 adenine nucleotides that is added to the 3' end of eukaryotic mRNAs
114
What is the constitutive mutant?
A mutation that results in the constant expression of a gene
115
Define polycistronic?
An mRNA sequence that codes for multiple proteins
116
What is peptidyl transferase?
The catalytic activity of the ribosome that forms peptide bonds during translation
117
What happens to the lac operon when adenylate cyclase is inactivated?
As cAMP would not be formed there would be no positive regulation
And so expression would be low even when glucose levels are low and lactose is high
118
What happens to the lac operon when there is a frameshift mutation near the start of the lacI gene?
This is likely to inactivate the lacI repressor and results in constitutive expression of the lac operon
119
Where does an activator protein bind in an operon?
In the promoter region
120
