1
Q
why did Mendel use pea plants to examine?
A
He used the pea plants as they had many characters to assess + all traits followed laws of Mendelian inheritance.
2
Q
btw just a cool side note xxx
A
Need to remember that, when talking about Mendel’s work, he didn’t know what chromosomes, genes, alleles, etc, were. He called everything ‘heritable factors’ and just formulated the basic laws of inheritance.
3
Q
describe 3 basic points in the history of genetics
A
- Mendel (1822-1884)- ‘The Father of Genetics’
- ‘Genetics’ coined by Bateson in 1905
- ‘Gene’ coined by Johannsen in 1909.
4
Q
name 5 processes in medicine + science that genetics is important for
A
cloning, stem cell research, genetic testing, gene therapy, DNA fingerprinting
5
Q
which 3 things did Gregor Mendel’s work lead to?
A
- the discovery of dominant + recessive traits
- the concept of the gene (‘heritable factor’)
- the formulation of the basic laws of inheritance
6
Q
what is a character?
A
a heritable feature of an individual e.g. flower colour, seed shape, stern length
7
Q
what is a trait?
A
a variant form of a character (the phenotype) e.g. purple, axial, tall
8
Q
what is Mendel’s first law- The Law of Segregation?
A
“the two forms of a gene (alleles) present in each parent segregate independently”
9
Q
Mendel formulated his first law by studying the results of monohybrid crosses- what are they?
A
a cross between 2 true-breeding (produce offspring identical to themselves) individuals differing in only ONE character e.g. colour
10
Q
how were monohybrid crosses such as this important for finding out about recessive traits
A
The important thing Mendel did was COUNT- he did the experiments for this multiple times + the average ratio he got was 3 green: 1 yellow. Always, in the F1 generation the yellow was lost + in F2, it reappeared.
Therefore, heritable factor for the recessive traits hadn’t been lost in F1, it was just masked by the dominant trait (green).
the same pattern of inheritance was seen for all 7 of the characters he studied
11
Q
what were the 2 conclusions Mendel came to after observing monohybrid crosses such as this?
A
- one trait is dominant (e.g. green pod) and the other is recessive (e.g. yellow pod).
- the heritable factor for the recessive trait hadn’t been lost in the f1- just masked by the presence of the factor for the dominant trait
12
Q
what were the 5 characteristics of Mendel’s model?
A
- variations in inherited characteristics are due to the existence of alternative versions of heritable factors called alleles
- for each character, an organism inherits 2 alleles, one from each parent
- if the 2 alleles differ, then the dominant allele determines the organism’s appearance (phenotype)
- the alleles don’t blend when present in the same individual- they remain discrete
- the 2 alleles segregate during gamete formation- and end up in different gametes.
13
Q
what does this picture show about how offspring receive gametes from parents?
A
each parent has 2 alleles- but only one is passed to an individual offspring via the gamete (pollen or egg)
each offspring receives one allele from one parent + the other allele from the other parent
14
Q
what is the phenotypic ratio of this cross?
A
3:1
15
Q
what do test crosses do + how would you carry one out?
A
determine whether a dominant-trait thing is homozygous dominant or heterozygous because we don’t know just by looking.
Just cross it with a homozygous recessive.
16
Q
what is Mendel’s second law- The Law of Independent Assortment?
A
“each pair of alleles (gene) assorts independently of each other pair of alleles (gene) during gamete formation”
17
Q
how did Mendel formulate his second law- The Law of Independent Assortment?
A
dihybrid crosses
18
Q
the ratio for all dihybrid crosses is 9:3:3:1, why not 3:1 like with monohybrid?
A
If independent assortment didn’t happen (linking), the gametes aren’t separated, and you would get the same 3:1 ratio. Instead, the yellow, green, round + wrinkled gametes are all independent of each other, so 16 combinations can happen, in a 9:3:3:1.
19
Q
how do you work out phenotypic ratios for crosses between 3 or more characters e.g. RrYyGg x RrYyGg?
A
you can use little Punnett squares for each trait to get individual probabilities of those, then multiply them together for specific stuff
20
Q
how were chromosomes first identified + by who?
A
Chromosomes were first identified under a microscope by Walter Sutton who extracted them from grasshoppers slay king; this supported Mendel’s work, which wasn’t loved by loads now.
21
Q
describe the stages of meiosis
A
22
Q
which 3 conclusions did Sutton come to that kinda supported Mendel’s theories?
A
- chromosomes occur in pairs in somatic cells.
- chromosome pairs segregate equally into gametes
- different chromosome pairs assort independently
he reasoned that if chromosomes carry genes, then this behaviour would explain Mendel’s observations
23
Q
which 2 things does the chromosome theory of inheritance state?
A
- Mendel’s ‘heritable factors’ (genes) are located at specific positions (loci) on chromosomes
- it is the chromosomes that undergo segregation + independent assortment
24
Q
why does independent segregation of chromosomes happen?
A
each allele is on a different homologous chromosome + moves to opposite poles in anaphase 1
25
how does chromosome behaviour in meiosis explain Mendel's Law of Independent Assortment?
explained by the random way that the homologous pairs of chromosomes line up on the metaphase plate during meiosis 1.
chromosomes can line up any which way along the cell, and the daughter cells from meiosis/ mitosis can have any random combination of alleles.
e.g. here, both possibilities are equally probable.
26
example of how chromosome behaviour in meiosis 1 leads to independent assortment of genes xxxxxx
fein
27
independent assortment of chromosomes means there's 4 possible combinations of alleles at 2 loci e.g. YR, yr, Yr, yR. are they all equal or not?
all equal
28
name 4 sources of genetic variability in sexual life cycles
1. mutations
2. independent assortment of chromosomes in meiosis 1
3. crossing-over between homologues chromosomes in meiosis 1
4. random fertilisation of ova by sperm
29
what are recombinant phenotypes (produced in dihybrid crosses)?
the combination of phenotypes looks different to the parents.
30
which processes in meiosis can recombinant phenotypes occur in?
1. independent assortment (for genes of DIFFERENT chromosomes)
2. crossing over (for genes of the SAME chromosome- the only way genes on the same chromosome can get different gene combos!!!!)
31
which part of meiosis does crossing over happen
during the pachytene phase of prophase 1
32
describe how crossing over happens (synaptonemal complex) + the resulting gametes after meiosis (3)
1. begins with synapsis- pairing of homologues
the synaptonemal complex is a cohesin-protein 'zipper' that holds homologues together in a tetrad.
2. this leads to crossing over, where the DNA is cut + re-fused together at the same specific gene loci, such that they swap genetic material.
3. Results in chromosomes that have 1 original parental sister chromatid + 1 recombinant sister chromatid.
You end up with 4 gametes after meiosis, 2 of them carrying parental chromatids only, and 2 carrying recombinant ones.
33
what is recombination frequency?
the percentage of the offspring that inherit a combination of alleles that differs from either parent
34
what is the equation for recombination frequency?
35
how are recombination frequencies estimated?
studying a test cross e.g. a cross between a double/ triple heterozygote and a double recessive line e.g. AaBb x aabb.
36
what will recombination frequency always be with genes on different chromosomes, due to independent assortment?
50%
37
what will recombination frequency be approximately with genes on the same chromosome?
more than 50% of the offspring will be parental, and less than 50% of the offspring will be recombinant.
(btw that table is wrong xxxx)
38
who was the first to observe gene linkage, and how did he do it?
T.H.Morgan- did experiments and cool stuff with vestigial wing mutants of Drosophila melanogaster (fruit flies)
39
what would be the results of this test cross between these epic fruit flies?????
40
calculate the recombination frequency of this cross
largest phenotypic class tells you what’s parental- use the numbers!!!!!
41
draw a coupling heterozygote and a repulsion heterozygote for epic fruit flies
42
how would you draw a chromosome linkage map from these results?
(btw may have noticed 9+9.5 doesn’t add to 17, well well well multiple crossing overs can take place. If we have those 2 crossing overs, we must underestimate the distance)
43
btw may have noticed that distances in chromosome linkage maps like these don't add up properly, why is that + what does it mean?
multiple crossovers lead to an underestimate of the distance between 2 loci, meaning recombination frequency above 50% aren't possible.
genes far apart on the same chromosome appear to assort independently.
44
calculate the individual recombination frequencies between b+cn, cn+vg, and b-vg using this weird method
45
how was sex linkage first observed in Drosophila (fruit flies), with the red eye and white eye stuff
explanation: the Y chromosome lacks the 'white' locus. the eye colour locus is on the X chromosome.
means that the male fly only needs 1 copy of the mutant X-white allele to have white eyes
46
what is incomplete dominance?
where a dominant allele doesn't completely mask the effect of a recessive allele at the same locus.g. flower colour in snapdragons
= diluted phenotype
47
what is co-dominance + how is it different from incomplete dominance?
where each allele affects the phenotype in separate, distinguishable ways
e.g. the ABO blood group system in humans. the I gene encodes an antigen found on RBCs surface.
=traits appear TOGETEHR + remain distinct, unlike incomplete dominance
48
what is pleiotropy?
where a single gene has multiple effects on the phenotype
e.g. in peas, the gene that determines flower colour also affects seed colour.
also, loads of diseases that are cased by single gene mutations have pleiotropic effects.
49
what is polygenic inheritance?
where a single trait is determined by multiple genes
e.g. a characteristic of polygenic inheritance is that the trait shows continuous variation in the population in like weight, skin, etc
50
what is epistasis?
when one gene masks or modifies the expression of another one
e.g. if this mouse is homozygous recessive at the C locus, it doesn't matter which alleles are present at the B locus- mouse will always be white
51
what are the results of a dihybrid cross when epistasis is involved, instead of 9:3:3:1, why?
1/4 of the mice are homozygous recessive at the C locus
52
what is the study of cytogenetics?
the study of structure and function of chromosomes
53
what is karyotyping?
a preparation of chromosomes arranged in size order
54
describe the process of like making a karyotype in the lab (6)
1. add some blood to a flask + phytohaemaglutinin to stimulate mitosis, then culture flask
2. incubate 2-3 days, then treat with colcemid for 1-2 hours to stop mitosis in metaphase
3. transfer cells to tube, then centrifuge them + add hypotonic solution to lyse cells
4. transfer to tube containing fixative
5. drop cells onto microscope slide + stain with Giemsa
6. examine under microscope + arrange into karyotype
55
why is Giemsa staining cool + needed?
you do most of the identification of the homologous pairs by length, banding pattern + centromere position, like in here you basically can’t tell.
56
in terms of centromere placement, what do metacentric, sub-metacentric, and acrocentric mean?
57
what are the 2 purposes of karyotyping?
1. detecting changes in chromosome number
2. detecting changes in chromosome structure
58
what is polyploidy in terms of chromosome change?
extra whole ahh sets of chromosomes e.g. instead of diploid (2n), could be triploid (3n) or tetraploid (4n)
59
how common is polyploidy in animals and plants, and why?
Polyploidy is quite common in plants but rare in animals, as gene expression is more closely controlled in animals. Animals are just way more complex, and sexual reproduction also goes into it.
Plants can propagate asexually, and in evolution, polyploidy happens more in plants so it’s kind of just there.
It’s also used in agriculture, like the cultivated banana obviously xxxx
60
what is aneuploidy in terms of chromosome change + what are the 2 forms?
some additional or missing random chromosomes
can be either monosomy (one chromosome missing) or trisomy (one extra chromosome)
61
how common is aneuploidy in animals + give an example of a disease it cause?
Aneuploidy is a bit more common in animals, happens in 50% of human conceptions e.g. trisomy 18 (additional chromosome 18– Edward’s syndrome, creates severe health problems due to the extra gene expression.
62
what is non-disjunction?
it causes aneuploidy. when chromosomes don't separate in meiosis 1 or 2.
63
if non-disjunction happens in meiosis 1, how many of the 4 gametes resulting will have trisomies or monosomies?
if it happens in meiosis 1, then there will be 2 trisomy’s + 2 monosomies,
64
if non-disjunction happens in meiosis 2, how many of the resulting 4 gametes will have trisomies or monosomies?
if it happens in meiosis 2, then there will be 1 trisomy, 1 monosomy + 2 normal daughter cells.
65
what is the only autosomal aneuploidy that permits living into adulthood?
Down's syndrome
66
aneuplodies of Down's syndrome are generally ok + way more common than autosomal ones, why?
like women already have 2 X chromosomes and usually one of them is inactivated in most of our cells (some not completely), so yeah if we get any extra X’s, they’re just inactivated.
67
name some characteristics of Down's syndrome (5)
1. characteristic facial features
2. short stature
3. some level of learning disability
4. heart defects
5. susceptibility to leukaemia + Alzheimer's
68
does incidence of Down's syndrome increase or decrease with maternal age?
increase
69
does Down's syndrome increase or decrease susceptibility to disease?
Although Down’s syndrome increases susceptibility for some diseases like Alzheimer's , it also decreases susceptibility for some like hypertension + some tumours- all to do with the genes expressed on c.21.
70
name 3 ways people can screen for chromosomal abnormalities with Down' syndrome
1. blood tests to detect specific proteins associated with a Down's foetus
2. ultrasound scans- measuring the size of the nuchal pad at the nape of the foetal neck associated with Down's syndrome
3. amniocentesis + karyotying
71
why is amniocentesis only done as like a last resort, and how is it done briefly?
Amniocentesis is often done if screening shows traits of a baby linked with Down’s syndrome, by withdrawing some amniotic fluid and creating a karyotype with that. There is a tiny risk of spontaneous abortion with that so only offered if there’s an increased risk. Nowadays we do some PCR tests for this + Edward’s.
72
Turner's syndrome is the only viable human monosomy, which chromosome is missing?
a lack of one of the sex chromosomes. Again we can tolerate this for reasons described.
73
what is the statistic for how often Turner's syndrome occurs?
1 in 2500 live female births
74
name some characteristics of Turner's syndrome (3)
1. phenotypically female
2. sterile due to lack of maturation of sex organs
3. oestrogen replacements therapy leads to development of secondary sex characteristics
75
Klinefelter's syndrome is one of the most common genetic abnormalities, which chromosome is gained?
either an extra X or extra Y whichever way you want to think about it- XXY. Even if you’re like XXXXXXXXY, you’re still essentially male because of the Y chromosome.
76
what is the statistic for how often Klinefelter's syndrome occurs in liv male births?
1 in every 500-1000
77
name the characteristics of Klinefelter's syndrome (4)
1. essentially male, but with some female characteristics
2. tall
3. sterile
4. treated with hormone replacement therapy (testosterone)
78
what is a chromosomal inversion (type of mutation)?
79
what is a reciprocal translocation (type of mutation)?
80
how would you name this little part of the chromosome here where the red arrow is pointing?
7p22.2
81
Cri-du-chat syndrome is caused by which chromosomal mutation + where in the chromosomes?
deletion somewhere in the 5p12.2 chromosome region.
82
what is the statistic for how often Cri-du-chat syndrome happens in live births?
1 in 50,000
83
what are the characteristics of Cri-du-chat syndrome (5)?
1. babies have cat-like cry
2. defects in glottis and larynx
3. wide face with saddle nose
4. physical + intellectual disability
5. range of severity, depending on extent of deletion
84
Prader-Willi syndrome is caused by a deletion in which chromosomal region?
15q1.12
85
what is the statistic for how often Prader-Willi syndrome happens in live births?
1 in 15,000
86
name some characteristics of Prayer-Willi syndrome + genomic imprinting (6)
1. poor suckling reflex in infants
2. uncontrollable eating in later life
3. obesity
4. diabetes
5. poor sexual development in males
6. only occurs when the affected chromosome is inherited from the father due to GENOMIC IMPRINTING
87
what is genomic imprinting?
the process that affects certain genes, whereby either the maternal or paternal copy of the gene is silenced
88
Angelman's syndrome is very similar to Prader-Willi's, how?
If you have a deleted chromosome 15 from the father= Prader-Willi syndrome. If you have a deleted chromosome 15 from the mother= Angelman’s.
(because the imprinted genes on the other chromosome are different in each case)
89
name some chracteristics of Angelman's syndrome (6)
1. happy demeanour
2. inappropriate laughter outbursts
3. intellectual disability
4. severe speech problems
5. stiff limb movements
6. seizures
90
familial Down's syndrome is caused by a translocation mutation, describe it
one normal copy of c.21, with c.14 having the other c.21 attached to it.
91
how many cases of Down's syndrome does familial Down's syndrome account for?
like 5%
92
how much chance is there of having a child affected with Down's syndrome in this case?
1:3 chance of having a second affected child
93
chronic myelocytic leukaemia (CML) is caused by a translocation, describe it
a 22-9 translocation. Small portion of the end of an arm off c.9 swaps places with quite a big portion off the end of c.22.
94
how many people are affected with CML?
1 in 50-100,000 of the population
95
is CML inherited or spontaneous?
spontaneous
96
95% of people with CML have the Philadelphia chromosome, what is that?
Philadelphia chromosome is just a truncated c.22.
97
translocation in the CML disease creates the BCR-ABL fusion gene, what is that + explain?
an oncogene that stimulates over-production of white blood cells.
the ABL gene is a tyrosine-kinase, which is activated when fused with the BCR one, which stimulates the cell cycle and gets it going, which is uncontrolled.
98
how many cases of leukaemia does CML account for?
15-20%
99
which age group is CML most common in?
middle aged/ elderly
100
Mendel, Darwin + Miescher are important people in major breakthroughs in biology for genetics, describe their discoveries + how they link with DNA discovery
Mendel- discovered dominant + recessive traits, concept of the gene, and formulation of the basic laws of inheritance.
Darwin- natural selection theory of evolution + all species of life have descended from a common ancestor over time.
Mieshcer- nucleic acid
DNA was discovered as a major chemical of the nucleus at about the same time Mendel was working out genetic inheritance and Darwin published his famous works on evolution.
101
describe Freiedrich Miescher's experiment for discovering nucleic acid (4)
1. WBCs were taken from pus from used bandages (from soldiers in the Crimean War ewwww)
2. nuclei in them was purified
3. extracted nuclei + found a precipitate rich in phosphorus (P) + nitrogen (N) = 'nuclein'
4. N-rich fraction= protein. acidic P-rich fraction= NUCLEIC ACID
102
earlier it was thought that proteins in chromosomes contained the hereditary info instead of DNA, describe + explain Griffith's experiment that disproved this
Griffith's experiment-
used 2 strains of Streptococcus pneumoniae- a benign (R) and a virulent (S).
R lacks a protective capsule + is destroyed by the immune system, S isn't.
then the mice thing happened (picture).
explanation: the heat-killed S form experiences this transforming principle (DNA) that transforms the R-form. the genetic material was capable of reprogramming R-form into S-form, disease-causing cells.
103
Avery, McCarty + Macleod built on Griffith's experiments, describe the results of their experiments and how they found out the transforming principle is DNA (6)
1. resistant to proteases- not protein
2. resistant to lipases- not lipid
3. resistant to ribonucleases- not RNA
4. ethanol-insoluble (precipitate formed + supernatant no longer transforming)- not carbohydrate
5. high molecular weight
6. positive reaction to the Dische test for the deoxyribose of DNA
104
Hershey + Chase did experiments to fully prove that DNA was the genetic material inside bacteriophages, describe them + the results
1. phage PROTEIN labelled with radioactive S
2. phage DNA labelled with radioactive P
phages allowed to infect a bacterium + the virus and bacteria were separated by centrifugation.
bacterial pellet has the radioactive P, but the S was in the supernatant.
conclusion- DNA injected into the cell, not protein
105
Chargaff was interested in the 4 bases of DNA, what is Chargaff's rule (3)?
the bases must contain genetic code as the sugar and phosphate groups were invariant.
1. ratio of the bases wasn't 1:1:1:1
2. ratio is species-specific
3. base composition always obeys A=T + C=G (main one)
106
describe what photograph 51 was
X-ray diffraction photo of DNA taken by Raymond Gosling (Rosalind Franklin's student)
107
which bases are purines + which are pyrimidines?
108
what were the 4-ish conclusions from the X-ray diffraction photo?
1. DNA is a helix
2. it's 2nm wide (width of 2 nucleotide chains)
3. length of each turn in 3.4 nm
4. distance between repeating units (nucleotides) is 0.34nm
so, there's 10 nucleotide pairs per turn
109
how did Watson and Crick realise that the bases would only fit in the DNA double helix if purines were paired with pyrimidines?
110
which bond is stronger, the one between A-T or C-G?
C-G.
111
how did Watson and Crick kind of discover how DNA replicates?
they suggested that each strand of DNA can act as a template for the synthesis of a new complementary strand- semi-conservative replication
112
Meselson + Stahl did an experiment proving semi-conservative replication, describe xxxx
1. bacteria cultured in medium heavy 15-N
2. bacteria transferred to medium with light 14-N
3. DNA sample centrifuged after 1st replication
4. all DNA of immediate density= 1 light strand and 1 heavy strand
113
describe the 'proof-reading' ability of DNA polymerase
the DNA can remove incorrectly inserted nucleotides
114
name the 3 things DNA polymerase requires to work
1. single-stranded template DNA
2. all 4 nucleoside triphosphates (dNTPs)
3. free 3' hydroxyl (primer)
115
how does DNA polymerase gain its energy?
from breaking phosphate bonds
116
where does replication of DNA begin?
origin of replication- a particular sequence in the genome where replication is initiated.
117
each replication fork has a leading + lagging strand, describe basically what they are
the leading strand is replicating continuously, whereas the lagging strand is replicating discontinuously forming short fragments
118
describe synthesis of the leading strand of DNA, and how the RNA primer, sliding clamp + DNA pol 3 factor in
119
describe synthesis of the lagging strand in DNA
1. a primase enzyme forms an RNA primer for the template strand attaches, and the lagging strand is formed in fragments called Okazaki fragments.
2. the primase detaches and reattaches behind the 1st primer + fragment, and makes another primer + fragment.
3. DNA pol 3 replaces RNA with DNA
4. DNA ligase joins Okazaki fragments
120
what is the difference between DNA polymerase 3 and DNA polymerase 1?
both in prokaryotes, similar proteins do the same job in eukaryotes.
3: it extends the DNA/ RNA strand from the 3' end, copying the template
1: removes the RNA primer + fills in the gaps between Okazaki fragments
121
Archibol Garrod was the first to connect inherited human disorders with Mendel's 1st law- Alkaptonuria is one such disease. describe what it is
when urine darkens from yellow-brown-black after exposed to air.
urine of alkaptonuria patients contains large amounts of homogentistic acid (originally called alkapton)
122
alkaptonuria (piss disease) is inherited in a mendelian fashion, is it autosomal dominant or recessive?
autosomal recessive
123
what were Archibald Garrod's hypotheses about Alkaptonuria (2)
1. alkaptonuria patients lack the enzyme necessary for breaking down homogentisic acid
2. lack of the enzyme due to a defect in a gene- we now know that the missing enzyme is homogentisic acid oxidase
124
what were the 3 conclusion from Garrod's alkaptonuria experiments?
1. defects in genetic material can lead to specific diseases, which can be inherited
2. Mendelian genetic inheritance can be observed in humans
3. lack of an enzyme is due to a defect in a gene
unfortunately, Garrod's conclusions were largely ignored at that time
125
Most higher organisms have diploid genomes: identifying new recessive mutations requires at least 2 generations of crosses as seen in Beadle + Tatum's 'one gene, one enzyme' experiments. Neurospora was an easy way to see defects in metabolic pathways as it's haploid so recessive traits appear in offspring. why else were neurospora good to test on (2)?
normal ("wild-type") Neurospora (bread mould) can grow without many nutrients because it has enzymes to generate its own e.g. amino acids
could easily use it to test by growing Neurospora on defined media which lacked one or more specific nutrients.
126
what was Beadle + Tatum's theory about neurospora?
Their theory was that if they damaged the DNA for one of these enzymes, then the cells would not be able to grow without the particular nutrient.
127
what did Beadle + Tatum's experiments with Neurospora entail like what did they do?
To test if genes were responsible for specific chemical reactions they irradiated Neurospora with X-rays (known to cause mutations).
128
describe the difference between the minimal + complete media here used in the Beadle + Tatum experiments on Neurospora
The complete medium basically just has everything the organism is going to need to grow. Minimal media don’t contain any added nutrients like none, so the cells put on the media need to be able to grow by themselves.
129
Beedle + Tatum found aa series of auxotrophic mutants after their experiments on Neurospora, explain what they are?
Auxotrophs are mutant strains that cannot synthesize a particular molecule required for growth (such as arginine) – therefore they will only grow if they are supplied with that molecule.
The experiments kind of determined that the species not growing must have a defect in the enzymes producing arginine.
130
after Beedle + Tatum identified auxotrophic mutants in their neurospora experiments, describe the process of how they screened them (5)
1. culture individual spores on COMPLETE medium
2. transfer to MINIMAL medium (MM) to identify possible auxotrophs
3. test candidates for growth on MM supplemented with different classes of nutrients (vitamins/ amino acids/ nucleotides)
4. test candidates for growth on MM supplemented with individual amino acids (or vitamins/ nucleotides)
5. identify the amino acid that allows your mutant to grow
131
Srb + Horowitz identified 3 classes of arginine auxotroph following the Beedle + Tatum experiments, here they are xxxxxxx
132
describe Beedle + Tatum's 'one gene- one enzyme' hypothesis
Beadle + Tatum proposed that each enzyme in a pathway was controlled by one gene.
Role of a gene is to encode an enzyme - and that for each enzyme there is a gene
133
what's the issue with Beadle + Tatum's 'one gene- one enzyme' hypothesis, and which hypotheses came from this issue?
not all gene products are enzymes.
As knowledge of proteins grew, it became clear not all proteins were enzymes. For example there are many structural proteins
Therefore:
‘One Gene – One Protein’
Further work showed some proteins are made of more than one polypeptide chain, for example haemoglobin is formed of alpha and beta polypeptides.
Therefore:
‘One Gene – One Polypeptide’
134
how do genes specify proteins?
can't do it directly
"Messenger” required – information transferred from nucleus to cytoplasm
Messenger is ribose nucleic acid (RNA)
135
describe the structure of RNA
a polymer of nucleotides containing ribose sugar and combinations of four bases: adenine, guanine, cytosine and uracil – usually single-stranded
136
Pulse-chase experiments provided evidence for mRNA, describe the experiments, and what they showed
With the experiments radioactively labelling RNA, the 15 min label shows that most of the uracil is in the nucleus, indicating it’s made there. After 90 minutes, it is shown that the RNA has migrated out of the nucleus into the cytoplasm.
Proved that RNA is made in the nucleus + moves into the cytoplasm
137
what does the central dogma mean?
DNA makes mRNA makes protein
138
what is transcription, and what is it catalysed by ?
Transcription is the synthesis of a mRNA molecule using one strand of the DNA as a template such that G pairs with C and A pairs with U
Transcription is catalysed by RNA polymerase
139
the 1st step of protein transcription is initiation, describe xxxxx
when RNA polymerase binds to the promotor region, the DNA is unzipped, and the RNA polymerase starts sliding along wow.
140
DNA polymerase requires a primer to work, does RNA polymerase?
Unlike DNA polymerase, RNA polymerase does not require a primer (does not need an existing 3’ OH group to add on to).
141
2nd step of protein transcription is elongation, describe xxxx
RNA polymerase moves along the DNA template, unwinding the double helix and catalysing the addition of ribonucleotides to the 3' end of the growing RNA molecule.
142
3rd + final step of protein transcription is termination, describe xxxxx
the termination sequence is transcribed into the RNA, and when the RNA polymerase comes across it, it dislodges the RNA strand
143
how many combinations of amino acids can be made from the 4 DNA bases?
4^3 permutations = 64 combos
(3 bases is 1 amino acid so cube 4)
144
Francis Crick and Sydney Brenner proved that “codons” are three letters, which was the first codon to be proved + how?
UUU, coding for phenylalanine was the first codon to be proved.
Took artificial mRNA consisting entirely of Uracil and added to a cell-free translation system.
Found that protein consisted entirely of phenylalanines.
Similar stuff was carried out for glycine, proline + lysine, + more complicated experiments then had to be performed to work out the remainder of the codons.
145
61 of the 64 codons code for amino acids…., what are the other 3 doing + identify them
UAA, UAG and UGA specify “stop codons” to signal the end of translation.
146
why is AUG a weird codon?
AUG has a dual role: the start codon and also encodes Methionine.
147
The Genetic Code is specific but redundant, what do both of these mean?
Each codon can only specify one amino acid: so it is specific.
But amino acids can be specified by more than one codon: so it is redundant.
148
The Genetic code is (almost) Universal, what does this mean, what does it suggest and why is it 'almost'?
Almost universal: the same codons are used for the same amino acids from bacteria to humans.
Suggests all life has common evolutionary ancestor.
A few rare exceptions:
Mitochondrial genomes, some stop codons
149
in translation of the triplet code, what does the resulting protein sequence depend on?
the reading frame
150
describe tRNA in terms of: length, single/ double stranded + shape?
Approx. 80 nucleotides in length
Single stranded but base pairs form within the chain G-C, A-U etc.
Clover leaf structure further folds to make L-shaped molecule
151
what is at either end of a tRNA molecule
anticodon (base pairs with codon)
amino acid attachment site- 3' hydroxyl group
152
why is it that tRNAs are specific for one single amino acid?
determined by its anticodon
e.g. tRNA with UAC anticodon (pairs with AUG codon) can only have methionine attached.
153
tRNAs are the adaptors between mRNA + amino acids, how do tRNAs become attached to the correct amino acid?
Specific attachment carried out by amino-acyl tRNA synthetases (acivating enzymes)
there is 1 for each of the 20 amino acids.
154
describe the 2-step attachment process of amino acyl-tRNA synthetases
the enzyme has binding sites for the tRNA, amino acid + ATP
1. ATP hydrolysed + amino acid joined to AMP (adenosine monophosphate)
2. correct tRNA binds + amino acid transferred from AMP to tRNA
155
where multiple codons specify for the same amino acid, which base usually differs?
last one
156
there's a slight issue with tRNAs, there's supposed to be a tRNA for each amino acid codon but there's 61 amino acid codons + only like 40 tRNAs, how do we get past this?
so yk how usually with multiple codons that code for the same amino acid, usually the last base differs e.g. leucine= CUU, CUC, CUA, CUG. there's this thing called wobble base pairing.
e.g. if the last base is a G, the 3rd base in the anticodon can be either U or C or something.
157
describe the basic structure of a ribosome (subunits, tunnels, what its composed of)
composed of rRNA + proteins
there's a large + small subunit, with an exit tunnel in the large subunit for the growing polypeptide to kind of grow out of
158
how many binding sites does a ribosome have + for what
3 tRNA binding sites:
A (amino acyl- tRNA binding site)
P (peptidyl-tRNA binding site)
E (exit site)
159
what do ribosomes do (include directions movement)?
catalyses stepwise formation of peptide bonds (amino acids added from amino (N) - carboxyl (C) terminus), moving in 5'-3' direction on the mRNA.
160
the 1st stage of protein synthesis is initiation, describe it (3-ish)
1. small ribosomal subunit binds to mRNA near its 5' end by recognising AUG start codon
2. initiator tRNA binds to AUG
3. large subunit binds so that the initiator tRNA fits into the P-site on the large subunit
(requires energy from GTP hydrolysis + proteins called initiation factors to stabilise tRNA + assemble ribosome)
161
the 2nd stage of protein synthesis is the elongation cycle, describe it (5)
1. incoming amino acyl-tRNA base pairs with codon in A-site (needs GTP hydrolysis)
2. peptide bond forms between NH2 of amino acid + COOH group of amino acid in P-site (catalysed by peptidyl transferase)
3. growing chain now in A-site
4. translocation- tRNA in P-site is ejected + the ribosome moves along the mRNA by 1 codon (GTP hydrolysis)
5. growing chain now in P-site + the A-site is free to accept next AA-tRNA.
162
what is peptide bond formation catalysed by in protein synthesis?
peptide transferase- an RNA enzyme (ribozyme)
163
the last stage of protein synthesis is termination, describe (5) xxxx
1. stop codon reaches A-site
2. there are no tRNAs for stop codons, so release factor enters A-site instead of AA-tRNA
3. water added to end of polypeptide chian
4. completed polypeptide released from tRNA in P-site
5. ribosome dissociates (2 GTP hydrolysed)
164
what are polyribosomes + why are they cool?
just a little cluster of ribosomes linked together
they increase efficiency of protein synthesis
they occur in eukaryotes + prokaryotes
165
how does the difference in eukaryotes + prokaryotes' nuclear membrane and organelles cause for difference in protein translation?
eukaryotes:
Nuclear membrane – mRNAs transported to cytoplasm before translation occurs.
Several different organelles – proteins must be trafficked to correct site.
prokaryotes:
No nuclear membrane – transcription and translation coupled
No organelles – proteins diffuse through cytoplasm
166
eukaryotes do this thing where they target proteins destined for secretion in the endoplasmic reticulum, describe how (6) xxx
1. polypeptide synthesis begins
2. an SRP binds to signal peptide, stopping synthesis for a bit
3. the SRP binds to a receptor in the ER, which forms part of a protein complex that as a membrane pore + signal-cleaving enzyme
4. SRP leaves, + polypeptide starts growing again, while moving across the membrane (signal peptide still on ER membrane)
5. signal-cleaving enzyme cuts of signal peptide
6. the rest of the completed polypeptide leaves + folds into final form
167
what is a DNA mutation + what are the 2 types?
permanent change in gene position/ number (chromosomal), or nucleotide sequence (point)
168
are mutations common?
no xxxx they're hella random
169
describe differences in mutations in somatic + germ-line tissue
somatic: NOT passed on to offspring, but passed to all cells descended from the original mutant.
~85% cancers caused by somatic mutations
germ-line: passed on to offspring. Cause inherited genetic diseases.
Raw material from which natural selection produces evolutionary change.
170
a base-pair SUBSTITUTION is a point mutation, what are the potential effects of it on the amino acid sequence? (3)
1. no effect- multiple codons code for amino acids
2. change in 1 amino acid- bad but not awful
3. forms a stop codon- very not good, no protein made
171
a base-pair INSERTION/ DELETION is an example of a point mutations, what are the potential effects on the DNA sequence?
1. frameshift- quite bad
2. forms a stop codon- very bad
3. insertion/ deletion of 3 nucleotdies- doesn't matter icl
172
apart from being spontaneous (e.g. base tautomerism), name some causes of mutation (5)
chemical: BASE ANALOGUES (molecules that sub normal bases for nucleic acids), MODIFYING AGENTS (chemicals that change the structure of bases), INTERCALATING AGENTS (insert themselves between bases)
physical: IONISING RADIATION (X-rays), UV
173
Spontaneous mutations can occur due to inherent instabilities in DNA e.g. base tautomerism, what is that?
Nucleotides can change to other conformations eg isomers and tautomers (at a low rate).
During DNA replication an incorrect base is inserted to form mismatched pair.
174
chemicals can resemble DNA bases but pair incorrectly incorporated in DNA which causes mutations, describe how this happens with 5-bromouracil
5 bromouracil is incorporated into DNA as though it were thymine
Once incorporated it tends to rearrange into a form that resembles cytosine.
Upon DNA replication, can result in a point mutation converting a AT bp to a GC bp.
175
chemicals can remove the amino group from adenine or cytosine which causes mutations e.g. nitrous acid/nitrite, describe how this happens with this example
deamination of cytosine to uracil, so replicated DNA containing deaminated C results in A being inserted.
daughter strand base pairing changed from CG to TA.
(Uracil DNA glycosylase has specific role in removing U from DNA to prevent mutations.)
176
chemicals can add hydrocarbon groups to nucleotide bases, which causes mutations (ALKYLATING AGENTS). describe how this happens with addition of an ethyl group at the O6 position
this alters the base-pairing characteristics
Results in a point mutation converting a GC to an AT.
177
intercalating agents can insert between bases + distort DNA helix, causing mutations, what effects would this have? (2)
Interfere with replication
Tend to cause frameshift mutations
E.g. ethidium bromide
178
ionising radiation can cause mutations in DNA, describe 2 ways how
1. directly ionises DNA or
2. ionises water to produce free radicals
this can damage bases + break double-strand
179
UV radiation can cause DNA mutations with pyrimidine dimers, describe how (3)
1. UV irradiation is absorbed specifically by the pyrimidine bases cytosine and thymine.
2. Covalent bonds can form between adjacent T or C nucleotides – pyrimidine dimers.
3. Blocks DNA synthesis leaving a gap opposite the site of damage.
180
thymine dimers in DNA can be removed by nucleotide excision repair (NER), describe how (4)
181
thymine dimers in DNA can be removed by nucleotide excision repair (NER), which disease is caused by deficiency of NER + what are the symptoms
Individuals with the rare hereditary disorder called xeroderma pigmentosum are deficient in NER.
XP characterised by development of skin cancer at an early age but only on those parts of body exposed to sun.
182
Cancer is a genetic disease caused by mutations, describe how mutations are linked to oncogenes + tumour suppresors
Mutations in two specific types of genes: oncogenes or tumour suppressors.
Most cancers occur due to mutations in somatic cells and are not inherited.
~15% cancers are caused by inherited mutations which give a pre-disposition to cancer (known as cancer predisposition syndromes). E.g. xeroderma pigmentosum.
183
describe the Ames test that used rat liver extract to determine if enzymes were in there that would convert non-mutagens to mutagens
184
how can single gene disorders be inherited, through autosomal or sex-linked genes? what does it depend on?
both, + can be dominant or recessive
depends which chromosome the gene is located on
185
give some examples of single gene disorders
Sickle cell anaemia (SCA)
Cystic fibrosis (CF)
Familial Hypercholesterolaemia (FH)
Huntington disease (HD)
Haemophilia
Duchenne Muscular Dystrophy (DMD)
186
how are single gene disorders caused (hint: mutations xxx)?
Caused by point mutations in single genes (as opposed to chromosomal mutations that affect many genes)
187
how common are single gene disorders?
Individually very rare but collectively affect 1-5% of population, costing NHS and social care services over £2 billion each year
188
describe the morbidity + mortality of single gene disorders in children
high xxxx
189
are single gene disorders curable?
Most currently incurable, although treatments improving.
190
when single gene disorders are inherited recessively, the phenotype is apparent only in individuals homozygous for disease allele. are the carriers affected, and how is gene function affected? what % do carriers have of having an affected child?
Heterozygous parents (carriers) unaffected, so disease is absent for most generations, but consanguinity (incest ew) increases frequency of affected people.
Complete loss of gene function leads to phenotype e.g. Cystic Fibrosis.
Carriers have 25% chance of having an affected child (Dd x Dd).
191
when single gene disorders are inherited dominantly, the phenotype is apparent in heterozygote parents. how is gene function affected?
Partial loss of gene function (haploinsufficiency) leads to phenotype e.g. Familial Hypercholesterolaemia.
loss of function is due to toxic proteins which interferes with cell function e.g. Huntington Disease.
192
CF is the most common example of a recessive single-gene disorder in Northern European Caucasians. what is the carrier frequency + how many people are born with it?
Carrier frequency: 1 in ~25
CF births: 1 in ~2500
193
if untreated, when will sufferers of CF die?
usually before 5, but now life can be extended into adulthood
194
Cystic fibrosis is a disorder originating in secretory epithelial tissue. what are some symptoms of CF?
1. accumulation of mucus in lungs, pancreas, digestive tract + other places.
2. effects like chronic bronchitis + recurrent bacterial infections.
195
what does the CF gene (CFTR) do? (btw CFTR = cystic fibrosis transmembrane conductance regulator)
it sits in the epithelial cells' plasma membrane + codes for a chloride channel, which regulates chlorine ions.
196
∆F508 is the most common mutation causing CF, what kind of mutation is it + what happens as a result?
Deletion of 3 base pairs (bp) results in loss of a Phe (F) residue at position 508. Protein does not fold normally and is more quickly degraded
There are >800 different CFTR mutations that cause CF.
197
how do defects in the CFTR gene cause CF?
Defect in Cl- transport causes extracellular mucus to become thicker and stickier.
198
describe briefly 2 easy treatments available for CF
life is prolonged by antibiotics and by daily massage to clear mucus from airways
199
why is sequencing of the chloride channel needed in giving medication for CF?
allows healthcare professionals to determine exactly what mutation a patient has. This information can then be used to offer suitable "precision medicines”.
200
drugs to improve function of the mutated protein in CF can help, depends on what effect the particular mutation has. give how gating mutations + others can be treated with drugs (2)
1. gating mutations can be treated with the drugs that help open gates.
2. Other mutations can be treated with combination therapies that bring more of the chloride channels to the surface and help those channels to operate more effectively.
201
how can gene therapy help CF?
1. gene therapy can provide patients with a copy of the correct chloride channel
2. clinical trials for integrating + non-integrating gene therapies are underway yayyyy
202
how many Afro-caribbean/ Afro-Americans does Sickle cell anaemia affects?
1 in 625
203
sickle cell anaemia is a autosomal recessive single gene disorder, what are 4 main symptoms?
anaemia
joint pain
swollen spleen
frequent severe infections
204
describe briefly the treatment available for sickle cell anaemia
Regular blood transfusions
205
describe a cure for sickle cell anaemia
Bone marrow transplant
206
how is sickle cell anaemia caused?
a single nucleotide substitution in β–chain of haemoglobin.
instead of glutamic acid (large, charged, hydrophilic side chain) being coded for, valine is (small, non polar, hydrophobic side chain).
Leads to incorrect folding of the protein.
The defective haemoglobin forms long chains of rigid polymers (after O2 is released) which deform the red blood cell.
207
give some pleiotropic effects of sickle cells (one gene having multiple phenotypic effects).
208
even though sickle cell anaemia is recessive (so the parent carriers don't show phenotype), they have a phenotype in low Oxygen conditions (‘sickle cell trait’). how is it linked to malaria?
they just have higher resistance for some reason lol
209
describe this autosomal dominant disease pedigree (sexes affected, parents, how much chance there is that an affected parent will have an affected child)
Males and females affected
Affected individuals have an affected parent
50% chance of affected parent having affected child
210
Huntington's disease is an autosomal dominant single-gene disorder + is characterised by late-onset degeneration of the brain. how many people are affected?
1 in 24,000
211
what are the main symptoms of Hungtington's Disease (4)?
1. jerky movements
2. personality changes
3. deterioration of walking, speaking and swallowing abilities
4. death will result from complications such as choking, infection or heart failure
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Huntington's Disease is a late-onset disease, what does this mean for when parents find out children have the disease?
(before genetic testing) HD sufferers would usually have had children before they knew they had the disease, already have been passed down
213
how has large gene mapping showed the cause of Huntington's Disease?
showed defect on chromosome 4.
HD geen contains some CAG repeats (because it encodes glutamine). 11-34 repeats is normal but people with HD have like 36-125.
214
Familial Hypercholesterolaemia (FH) is an autosomal dominant single-gene disorder. what are the symptoms? (3)
High levels of cholesterol in the blood from an early age
Cholesterol deposits build up in joints
Cardiovascular disease
215
describe briefly how Familial Hypercholesterolaemia can be treated easily (2)
Cholesterol-lowering drugs e.g. statins
Low cholesterol diet
216
Familial Hypercholesterolaemia is caused by lack of low density lipoprotein (LDL) receptor. compare what happens to blood if you have the LDL vs if you have FH.
normal: efficient removal of cholesterol from blood. LDL binds to + carries cholesterol in blood stream.
FH: cholesterol accumulates in the blood.
217
Familial Hypercholesterolaemia is an example of incomplete dominance, meaning heterozygotes can be affected but not as much as homozygotes. how much cholesterol can accumulate in the blood with both?
Homozygotes (hh) have a 6-fold increase in blood cholesterol - heart attacks at age of 2.
Heterozygotes (Hh) have a 2-fold increase in blood cholesterol - heart attacks by age of 35.
218
what is the frequency of homozygotic + heterozygotic familial hypercholesterolaemia births?
hh – 1 in 1 million births
Hh – 1 in 500 births
219
what is the dominant familial hypercholesterolaemmia caused by?
haploinsufficiency
220
Sex-linked conditions are caused by a gene on the X chromosome. if a carrier female + normal male procreate, what are the chances they'll have an affected daughter + affected son?
221
describe this pedigree of a sex-linked recessive trait (which sexes are carriers + affected, chance of female carriers passing down the disease to children, and chance of affected males transmitting to children) (3)
1. Females are carriers, mostly males affected
2. Carrier female will transmit to 50% of sons (affected) and to 50% of daughters (carriers)
3. Affected male cannot transmit to sons but will transmit to 100% of daughters (carriers)
222
Haemophilia is a sex-linked recessive, single-gene disorder. what are the symptoms (3)?
it's a blood clotting disorder so:
1. uncontrolled bleeding
2. tendency to extensive bruising
3. bleeding into joints
223
there are 2 types of Haemophilia, A + B. describe the mutations causing both + what is the incidence for both?
Haemophilia A:
Mutation in the gene for Factor VIII (on X chrom.). Incidence: 1 in 5,000 males
Haemophilia B:
Mutation in the gene for Factor IX (on X chrom.)
Incidence: 1 in 30,000 males
224
pedigree of haemophilia B in Queen Victoria's family xxx
225
Duchenne muscular dystrophy is a sex-linked recessive single-gene disorder, what is the incidence?
The most frequent lethal childhood genetic disease (1 in 3500 males)
226
how is Duchenne muscular dystrophy caused?
Affected gene codes for a muscle protein called dystrophin.
Dystrophin gene is very large (2.6 million bp – largest known human gene) – makes it prone to rearrangements
227
describe how Duchenne muscular dystrophy is caused by the Dystrophin gene being disrupted
1. Dystrophin is part of a protein complex that connects the cytoskeleton of a muscle fibre to the surrounding extracellular matrix through the cell membrane.
2. Muscles in DMD affected males progressively die. Death occurs by age 20 from respiratory or cardiac failure.
228
is Duchenne muscular dystrophy curable?
Currently incurable although there are promising gene therapy techniques under research to replace a normal copy of Dystrophin yayyyyy
229
how much did it cost to sequencing a human genome in 2003, 2014 + 2023?
2003: $100 million/genome
2014: $500/genome
2023: $100/genome
230
there is currently an increasing need for bioinformatics, which 4 things can we use computer technology for?
1. collect biological data
2. store biological data
3. analyse biological data
4. disseminate (spread) biological data
231
where in the central dogma sequence does gene expression happen (genes turned on/off)?
in the RNA-protein bit
232
Omics studies allows scientists to investigate large number of genes at once: genomics, transcriptomics + proteomics, what is the input type for all + give some examples of each e..g mass spec
233
give 2 scenarios in which omics studies are useful?
Diagnose suspected genetic disorders
Determine cancer mutations
234
The rapid increase in genomic and transcriptomic data is largely driven by advances in what?
Next Generation Sequencing (NGS) methods e.g. Sanger sequencing, Illumina (example of short-read sequencing) + Nanopore (example of short-read sequencing)
235
Omics terminology often reflects sequencing technology or input sample type/processing, name 2 types of sequencing technology + 2 types of input sample types
Different sequencing technology
1. Sanger sequencing
2. Next generation sequencing (Short-read sequencing + long-read sequencing)
Different input sample type/processing
1. DNA (Whole-genome sequencing, Exome sequencing)
2. RNA (RNA-sequencing
Single-cell RNA-sequencing
Spatial transcriptomics)
236
what is a digital read?
digital output of the sequencer, gained from the physical DNA/RNA nucleotide fragment studied.
237
where are the sequence ID + description of this FASTA format read?
Sequence ID= red bit, description= blue
238
FASTQ is a format specialised for storing raw sequencing data + more common although not 100% accurate so a quality line is needed for each nucleotide. label it here
epic pink line
239
after you receive digital reads from a sequencer, what do you do next (2)
Stitch the reads together like a jigsaw puzzle, then compare them to existing sequences (search databases containing sequences reported by others, + align to well-curated reference genomes).
240
Aligning reads to reference genomes allows you to detect genetic variation and quantify gene expression/presence, how do you do it with a reference sequence + why do you need it?
the reference sequence acts as a kind of map, so other reads people do can all be compared.
Always need a reference sequence as genes may start at different positions e.g. Gene A may start at position 3, so we need a reference to accurately compare.
241
with digital alignment, what does it mean if you have some reads that align with e.g. Gene A, and none with Gene B (in terms of expression)?
Gene A has been expressed + Gene B hasn’t
242
There is a 0.1% difference in the genome sequence between 2 individuals (roughly 1 in every 1,000 bases). Most genetic variations don’t have a functional impact. Why?
many codons code for the same amino acid
243
what is GWAS?
genome-wide association studies
244
how can we test if a genotype is associated with a trait?
Difference in frequency of a genotype between control and test group (stats test needed like Chi square or something)
245
why does GWAS help the process of investigate genome variants become more efficient?
as DNA makes RNA makes protein, any genes that don’t code for proteins we can just throw out (introns)- don’t have to study every variant.
Variants close to each other tend to get inherited together , so info can be captured by a subset of loci.
246
what else gives us additional insights to genome pattern in bioinformatics?
evolutionary conservation
247
how does linking DNA variation and gene expression give us additional insight to genome reads?
expression quantitative loci (qQTL).
e.g. people with certain DNA variation may show higher/ lower level of RNA
248
RNA-sequencing (RNAseq) can be used to study gene expression across the whole transcriptome. Gene expression may reflect... (5)
1. Cell type specialisation
2. DNA damage (for example UV exposure)
3. Pathogen
4. Drug treatment
5. Cancer prognosis
249
do genes usually work in isolation or with others?
Genes don’t usually work in isolation– they interact with each other in networks/pathways
250
Transcriptomics is getting smaller and bigger: from bulk RNAseq to single-cell RNAseq and spatial transcriptomics. Historically, how was RNAseq done on cells + how is it evolving?
Historically RNAseq is done as bulk (the tissue), but we realised that RNA come from a mixture of cells.
In the last 5 years or so, single-cell RNAseq and spatial transcriptomics took off.
Single cell RNAseq helps with this.
251
what does single cell RNAseq do (basic process with time frames)?
It captures the transcriptome for individual cell.
Barcoded gel beads get shot through this thing and get joined with a cell, producing single cell GEMs
~10,000 cells per experiment
- 2 days to run the experiment
- 3 months to do the bioinformatics analysis
252
what do spatial transcriptomics give you information on + 2 ways it works?
specifically where genes are expressed
1. Array based (~50µm per spot; a cell is typically 10µm)
2. High-plex in situ hybridisation (single-cell, up to 5,000 genes)
253
Complement data allows you to combine multiple omics data e.g. genomics + transcriptomics (multiomic/ multimodal projects). Why is this needed?
When you look at just one omics, sometimes you can miss key information.
You can also combine multiple types of large data e.g. transcriptomics + MRI scan + blood results- saves a lot of money for researchers as they can share experiments and stuff.
254
what does DNA barcoding allow? (looks like a barcode because they colour A, T, C and G differently)
species identification through a short section of DNA (amplified by PCR and then sequenced)
255
does DNA barcoding work for genes regions that are similar within the same species or distinct across different species?
both
256
give 3 real examples of where DNA barcoding is used
Food fraud detection
Identifying pollens
Pathogen surveillance
257
which sequences is the FASTA format good for and basically nothing else? lol (2)
nucleotide/ amino acid
258
give a virus who's evolution was studied with phylogeny
SARS-CoV-2
259
why is sequence alignment needed + give an example of an alignment file?
to make phylogenetic trees- CLUSTAL file format is a type of alignment file (.aln)
260
what is the website GeneCards used for + what does it do?
a human gene database with extensive information on each gene.
it collates information from different databases and provides links to the sources.
261
PDB (Protein Data Bank) format is used to do what.....
store information on protein structure (.pdb)
262
what can be retrieved from biological databases?
gene + protein information
263
what is the website BLAST used for?
searching databases for similar sequences to your query sequence
264
describe 3 reasons why bacteria are very easy to use when studying genetics + name a species commonly used
1. easily cultured (picture)
2. short generation time of like 20-30 mins- if mutations happen, you can see it immediately
3. haploid- if it were diploid, then the mutation will be masked by the wild-type form of the gene.
265
E. coli DNA is compacted in the nucleoid which occupies a large fraction of the cell volume, in a single chromosome which is the whole genome of E. coli. describe what it is (strands, number of base pairs, etc) + how it is different to other bacterial chromosomes
The E.coli chromosome is a single double stranded circular DNA molecule 4.6 million base pairs in length, and contains around 4500 genes.
Most bacteria have just a single circular chromosome.
266
Bacteriophages are viruses that infect bacteria; they are structurally and functionally diverse.
name the ones pictured here (if you can not really important)
267
why do we use bacteriophages and such to assess how recombinant bacteria are formed?
Bacteria aren’t like multi-cellular organisms and don’t go through typical sexual reproduction, so we can't use that lol
268
lytic (virulent) + temperate (lysogenic) bacteriophages have different life cycles sort of, lytic ones multiply and then lyse the bacterial cell releasing progeny bacteriophage particles. describe the lytic bacteriophage life cycle
1. T4 phage uses its tail fibres to stick to specific receptor sites on the outer bit of E.coli cell
2. the tail sheath contracts, thrusting a hollow core through the cell wall + membrane, phage injects its DNA
3. empty capsid of the phage is left as a 'ghost'. cell's DNA is hydrolysed
4. the cell's metabolic machinery, directed by phage DNA, produces phage proteins + nucleotides from the cell's degraded DNA are used to make copies of the phage genome.
5. phage parts come together, 3 separate sets of proteins assemble to form phage heads, tails + tail fibres
6. phage then directs production of lysozyme, an enzyme that digests the bacterial cell wall. with a damaged wall, osmosis causes the cell to swell, and finally to burst, releasing 100-200 phage particles.
269
lytic (virulent) + temperate (lysogenic) bacteriophages have different life cycles sort of, temperate ones can integrate into the bacterial chromosome and remain dormant, replicating along with the bacterial DNA. describe the temperate bacteriophage life cycle
1. occasionally a prophage exits the bacterial chromosome, initiating a cycle.
2. phage DNA circularises + integrates into the bacterial chromosome, becoming a prophage.
3. the bacterium reproduces normally, copying the prophage + transmitting it to daughter cells.
4. many cell divisions produce a colony of bacteria infected with prophage
270
Genes can be transferred between bacteria and genetic recombination produces new bacterial strains. mutations form new alleles, but recombination also does, how (definition)?
recombination is defined as the combining of DNA from two individuals into a single genome.
271
Gene transfer and recombination occurs through three processes: transformation, transduction (generalised, involving the lytic cycle and specialised involving the temperate cycle) + conjugation. describe the 1st step, transformation
uptake of naked DNA, where bacterial DNA from the cell gets put into the normal DNA basically lol
272
Gene transfer and recombination occurs through three processes: transformation, transduction (generalised, involving the lytic cycle and specialised involving the temperate cycle) + conjugation. describe the 2nd step, transduction of lytic life cycles
transduction is transfer of bacterial genes from one bacteria to another by a bacteriophage.
1: generalised- phage infects bacteria, host DNA is hydrolysed + phage DNA and proteins are made. occasionally a bacterial DNA fragment is packaged in a phage capsid. then, CROSSING OVER- transducing phages infect new host cells, where crossing over can occur.
the recombinants have genotypes (A+B-) different from either the donor (A+B+) or recipient (A-B-).
273
Gene transfer and recombination occurs through three processes: transformation, transduction (generalised, involving the lytic cycle and specialised involving the temperate cycle) + conjugation. describe the 2nd step, transduction of temperate life cycles
transduction is transfer of bacterial genes from one bacteria to another by a bacteriophage.
2: specialised- bacterial cell already has prophage integrated between genes A + B. occasionally, prophage DNA exits incorrectly, taking adjoining bacterial DNA with it. phage particles carry bacterial DNA (here, gene A) along with phage DNA.
then, CROSSING OVER- transfusing phages infect new host cells, where recombination (crossing over) can occur.
the recombinants have genotypes (A+B-) different from either the donor (A+B+) or recipient (A-B-)
274
Gene transfer and recombination occurs through three processes: transformation, transduction (generalised, involving the lytic cycle and specialised involving the temperate cycle) + conjugation. describe + explain the 3rd step, conjugation.
conjugation is the ability to form sex pili and to swap DNA by conjugation is determined by a plasmid called an F (for fertility) factor.
the sex pili are baso just a mating bridge that break after the DNA has been passed on
Here:
1. the F factor replicates in synchrony with the bacterial chromosome.
2. the F factor replicates in such a way that one end of the DNA molecule passes through the cytoplasmic bridge into the recipient cell where it circularises.
3. The recipient cell is called an exconjugant (F-). The donor keeps a copy of the F factor.
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some examples of bacteriophage conjugation xxxxx
In that last cheeky example, the B+ from the donor combines with the B- from the other one, so that’s why there is that weird B-+ there xxxx
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what are the terms for how genes are expressed when they code for proteins that are needed all the time, vs only required sometimes?
Genes which code for proteins required all the time by the bacterial cell are constitutively expressed
Other genes are only active (expressed) when they are required – regulated genes
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Gene expression in bacteria is often controlled at the level of initiation of transcription, when does bacterial transcription begin?
Transcription begins when RNA polymerase binds to a promoter.
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here, tryptophan (an amino acid) is absent. so what does this mean for the transcription repressor (made by a regulatory gene) + operon and what is this type of regulation called?
Negative regulation – binding of repressor/tryptophan to operator blocks transcription as repressor is active + operon is off.
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lactose metabolism is controlled by regulated synthesis of inducible enzymes. here, lactose is absent, what does this mean for the transcription repressor
+ operon?
lactose is absent, so repressor is active + operon is off
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lactose metabolism is controlled by regulated synthesis of inducible enzymes. here, lactose is present, so what does this mean for the transcription repressor
+ operon?
lactose is present, so repressor inactive + operon on
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negative regulation when it comes to expressing bacterial genes is when binding of a protein to an operator prevents transcription. describe how this happens with the trp operon + lac operon
1. trp operon, expression is off when tryptophan binds to repressor which then binds to the operator.
2. lac operon, expression is off in the absence of lactose when the repressor binds to the operator.
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positive regulation when it comes to expressing bacterial genes is where binding of a molecule to the operator turns on gene expression, using a cAMP receptor protein. describe how it works when lactose is present, and glucose is SCARCE or ABUNDANT. how do cells sense cAMP conc?
1. lactose is present, glucose scarce (cAMP high), abundant lac mRNA synthesis
2. lactose is present, glucose abundant (cAMP low), little lac mRNA synthesis.
The cell senses the concentration of cAMP via a receptor, not actually the concentration of glucose.
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compare eukaryotes + prokaryotes by: whether they're unicellular/ multicellular, whether they have membrane-bound organelles, and how many genes they have (approx)
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do prokaryotes + eukaryotes have small or large genomes respectively?
pro= small genome
eu= large genome
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comparative thing showing different genome sizes xxxxx
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give 2 reasons why eukaryotic genomes are generally so large?
1. The number of genes- more genes= greater complexity
2. The amount of non-coding DNA- in eukaryotes, 97% of DNA doesn't encode for for proteins/ RNA.
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non-coding DNA consists of...... (3)
1. Gene regulatory sequences (e.g. promoters)
2. Introns (non-coding sequences within genes)
3. Sequences of no known function (including repetitive DNA sequences – ‘Junk’ DNA?)
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Eukaryotic genes can be very large (regulatory sequences + transcribed region containing introns). describe what is meant by promoter, exon + intron....
Promoter: the part of the gene that controls its transcription
Exon: transcribed sequence that is represented in the final mRNA
Intron: intervening sequence in the transcribed region that is not represented in the final mRNA
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repetitive DNA sequences are made of interspersed repetitive DNA + tandemly repetitive (satellite) DNA. describe what interspersed repetitive DNA is, around how many bps it is and around how much of the genome it makes up
interspersed DNA is composed of repeated units scattered throughout the genome (about 100-10,000 bp).
copies aren't necessarily identical, but closely related.
makes up about 25-40% of most mammalian genomes e.g. Alu elements (300 bp repeats) make up 5% of the mammalian genome
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interspersed DNA sequences are kind of related to viral sequences, which could cause mutations evolutionarily. Don’t do that anymore though as they’ve been selected against.
Now we have Alu elements, how are they different
Alu elements are scattered around, but don’t jump around and stuff.
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Barbara McClintock won the Nobel Prize for discovering mobile genetic elements btw xxx (Alu elements)
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interspersed repeats in DNA can be detected in human chromosomes using FISH (Fluorescent in situ hybridization). what does the green fluorescence hear represent?
Green fluorescence shows location of Alu repeat sequences around the human genome
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repetitive DNA sequences are made of interspersed repetitive DNA + tandemly repetitive (satellite) DNA. describe how many bps regular satellite, mini satellite and micro satellite DNA's are. also, where is satellite DNA located + which genetic disorder is caused by abnormally long stretches of a repetitive sequence xxx (3)
they're usually 5 bp repeats like GTTAC
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centromere satellite DNA + telomere satellite DNA are important separately in what? (2)
Centromere satellite DNA is important in organisation of chromosomes and stuff. Telomere satellite DNA is important in protecting the ends of chromosomes from degradation.
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what is chromatin made of? (2)
protein + DNA
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why must DNA in a cell be packed in an organized manner?
to be accessible for transcription and replication
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describe the difference between heterochromatin + euchromatin
Heterochromatin: highly condensed during interphase, not actively transcribed
Euchromatin: less condensed during interphase, able to be transcribed
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during interphase, is most of the DNA in heterochromatin or euchromatin form?
During interphase, a lot of the DNA is in the euchromatin form, some of it is in heterochromatin which blocks transcription.
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what are histone proteins?
proteins with positively charged amino acids that bind to the negatively charged DNA
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around how many bps of DNA are wrapped around each histone octamer
like 200
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describe the process of how packing of DNA around histones forms chromatin
1. nucleosome bead of histone proteins binds to DNA strand (also histone H1 attaches to the strand as well)
2. forms like a 30 nm chromatin finer all packed together in a box thing
3. looped domains of the 30 nm chromatin fibre start forming like looping up + down (euchromatin)
4. during mitosis/ meisosis, those loops fold further up + down to form normal chromosome shape to form supercoil with 300nm diameter (heterochromatin)
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there are over 200 different cell types in the human body, each cell expresses which percentage of their 20,000 genes at any given time?
3-5%
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In both eukaryotes and prokaryotes, genes are continually being turned on and off in response to signals from their internal and external environments
in multicellular eukaryotes, there is long-term control of gene expression during which process?
cellular differentiation
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name some stages of the steps of a protein being formed from a gene that regulation of gene expression can happen
e.g. DNA unpacking= euchromatin/ heterochromatin. Host transcriptional regulation of the nucleus. mRNA degradation control= amount of protein produced, etc
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which level, transcriptional or translational, does most gene regulation happen, and why?
Most regulation of gene expression occurs at the transcriptional level, presumably because it is more energy-efficient (ie energy is not wasted making a mRNA that is not translated).
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gene expression can be coarsely adjusted by chemically modifying chromatin, with either DNA methylation (associated with gene silencing) or Histone acetylation (associated with gene activation). describe DNA methylation
Attachment of methyl groups (-CH3) to DNA bases- triggers formation of a compact chromatin structure.
Associated with inactive DNA- e.g. in cell types where a gene is not expressed.
Accounts for genomic imprinting in mammals.
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gene expression can be coarsely adjusted by chemically modifying chromatin, with either DNA methylation (associated with gene silencing) or Histone acetylation (associated with gene activation). describe histone acetylation
Attachment of acetyl groups (-COCH3) to histones
Acetylated histones grip DNA less tightly
Acetylation/deacetylation is involved in switching genes on and off
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changes in DNA methylation and histone acetylation affect chromatin structure. describe if DNA is methylated or not, and if histones are acetylated or not, when chromatin is closed
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changes in DNA methylation and histone acetylation affect chromatin structure. describe if DNA is methylated or not, and if histones are acetylated or not, when chromatin is open
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what are the 3 different RNA polymerases?
1. RNA polymerase I (pol I): ribosomal RNA
2. RNA polymerase II (pol II): messenger RNA (mRNA)
3. RNA polymerase III (pol III): small RNAs e.g. tRNA
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DNA promoters determine what... (2)
Determine where the transcription of the gene is initiated
Determine the rate of transcription
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Transcription begins when the RNA polymerase binds to a promoter. the TATA box is key here. describe it (3)
1. A key part of the promoter
2. Provides the site of initial binding of the transcription initiation machinery
3. Located 10-35 bp upstream of the transcription start site
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Before transcription can start, a preinitiation complex must form- RNA polymerases cannot bind to promoters on their own. Transcription factors are key here. describe the 4 key steps that occur in forming a preinitiation complex
1. Binding of TFIID: includes the TATA-binding protein (TBP) + TATA-associated proteins (TAFs)
2. Sequential addition of other ‘general transcription factors’ – first TFIIA and TFIIB
3. binding of TFIIF + RNA polymerase 2
4. followed by TFIIE +TFIIH-
to form the preinitiation complex
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in eukaryotes, why does transcription usually form low levels of gene expression?
The interaction between the transcription initiation complex and the basal promoter is very inefficient
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transcription-level gene expressions regulated by ‘specific transcription factors’ (‘activators’ or ‘repressors). what do they bind to? (2)
‘proximal control elements’ and ‘distal control elements’ (groupings of which are called ‘enhancers’)
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How do enhancers work when they may be 1000s of bps from the promoter?
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describe the modular structure of specific TFs (different domains), and what the transactivation domain is responsible for
they have specific domains that bind to DNA + specific domains that bind to proteins (transcriptional activation).
The transactivation domain is responsible for recruiting other proteins into the transcription factor complex .
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what are glucocorticoid receptors?
they're a type of nuclear receptor that play a crucial role in mediating the effects of glucocorticoids, which are steroid hormones like cortisol.
they regulate gene expression well xxxx
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describe how glucocorticoid receptors regulate gene expression
1. In the nucleus, the activated glucocorticoid receptor binds to specific DNA sequences called glucocorticoid response elements (GREs). It binds binds to DNA with zinc atoms called a zinc finger receptor I think.
2. This binding can either promote or inhibit the transcription of specific genes, leading to changes in protein production.
3. These changes can influence various bodily functions, such as metabolism, inflammation, and immune responses.
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why is polyadenylation important in post-transcriptional regulation?
stabilises mRNA.
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alternative RNA splicing allows 1 gene to code for more than 1 protein?
Regulatory proteins control intron-exon choices by binding to regulatory sequences within the primary transcript.
Different mRNAs can be generated in different cell types.
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what does the alpha-tropomyosin gene do in terms of gene expression in proteins?
The alpha-tropomyosin gene specifies different forms of the protein in different types of muscle
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The Dscam gene can generate >30,000 different proteins through alternative splicing. where are Dscam proteins located + how do they regulate brain development?
Dscam proteins are located on the surface of a growing neuron
Provide a cell recognition mechanism that regulates brain development
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describe what cell determination is + the appearance of a cell while it's in this stage
the process that leads up to the observable differentiation of a cell.
the cell doesn’t look any different to an undifferentiated cell (phenotype isn’t different), just when it’s committed to differentiation but hasn’t done it yet.
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Differentiated cells express different sets of genes, all cells express what we call housekeeper genes, but some express like cool specific ones. what do muscle cells express that most don't?
muscle cells express myosin
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The differentiated state of a cell is achieved mainly through transcriptional regulation – involving gene cascades. describe what that is and what it doesxxxx
Gene 1: master regulator gene. When activated, it produces transcription factors that will activate to promote expression of genes 2, 3 + 4, and so on.
That signal turns on Gene 1 (cell is determined). Cell-specific genes are then transcribed + produce proteins that change the cell’s appearance, function + behaviour.
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muscle cells develop from which kind of cells with the potential to develop into a number of different cell types???
embryonic precursor cells
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describe the process of determination/ differentiation of muscle cells from the point when they develop from embryonic precursor cells (4)
1. signals from other cells lead to activation of the myoD master regulatory gene encoding the MyoD transcription factor.
2. Cell determination has now occurred and the cells are called myoblasts.
3. MyoD activates the expression of other muscle-specific transcription factors.
4. These in turn activate genes for muscle proteins (such as myosin) and block cell division – non-dividing myoblasts fuse to form muscle fibres (REMEMBER!!!- muscle cells are non-dividing and then kind of fuse together, so cell division needs to not happen)
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why is myoD a good master regulatory gene for muscle cells?
Based on external signals those precursor cells receive, they turn on myoD. Once cell has been determined, it’s still going to like keep getting that signal, so there needs to be a way for the cell to not revert from a muscle cells. myoD maintains itself, helping this to happen.
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the 3 main phases of Drosophila development are establishing main axes, establishing segments + filling in the details. describe what these mean AND with time ranges
1. establishing axes e.g. anterior (front), posterior (back), dorsal (up), ventral (down).
In the 1st 4 hours after egg fertilisation, the nuclei divide but there’s no cell division, so cell’s basically just a bag of nuclei (syncytial blastoderm). Here, the main axes of the fly are established.
2. establishing head (3 segments), thorax (3 segments), abdomen (9 segments)
Around 4 hours after, the nuclei migrate to the boundary + cell walls form.
3. building various organs of the animal (wings, legs, eyes, etc).
3rd phase is basically just getting details of the segments like what will each of them be, e.g. thoracic segment has 2 legs, or something.
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this is a cool normal larva. how was the Bicoid mutant of this different physically?
In the Bicoid mutant, all the thoracic + head segments were missing. Obviously didn’t develop past that stage bless. As this was a mother I think, it resulted in embryos that didn’t develop properly + looked like that.
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Bicoid is an example of an ‘Egg-polarity’ gene. These determine the polarity of the egg – and therefore the fly. Encode proteins or mRNAs that enter the egg while it is still in the ovary. what happens when the maternal genes is defective?
When the maternal gene is defective, the eggs fail to develop normally (therefore called ‘maternal effect’ genes).
Encode transcription factors which initiate a cascade of gene activations
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A gradient of the Bicoid mRNA is already established in the unfertilized egg. which gradient does this lead to in the early embryo?
protein gradient.
That dark bit on the blob shows the Bicoid mRNA at the anterior end. Thia stage shows the thing still in its ‘bag of nuclei’ phase. Results in a gradient of Bicoid in the early embryo.
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which of the Bicoid drosophila axes: posterior, anterior, dorsal or ventral, does the Bicoid protein gradient determine in the fly? how?
anterior end.
Established the principle that a gradient of molecules (morphogens) can determine polarity and position.
Bicoid and other morphogens are transcription factors which initiate a cascade of gene activations.
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Egg polarity genes like Bicoid initiate a gene cascade that regulates Drosophila development. describe the 3 classes of segmentation gene that the gene cascade goes through, in between egg-polarity genes + the ending homeotic genes.
336
a gene cascade regulates Drosophila development: egg-polarity genes -> gap genes -> pair-rule genes -> segment polarity genes. describe what each stage determines/ establishes
1. EP genes: determine anterior-posterior axis and induce...
2. Gap genes: sub-divide the embryo into broad areas and induce...
3. pair-rule genes: establish pairs of segments and induce...
4. SP genes: establish the anterior-posterior axis of each segment and induce homeotic genes.
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Mutations in Gap genes produce a phenotype where part of the embryo is missing e.g. Krϋppel. which part is missing with Krϋppel mutants?
area of gene action
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there are quite a few homeotic mutants of Drosophila. what's the difference physically between wild-type ones + the ultrabithorax (mutated) ones?
3rd segment= 2 legs + 2 halteres usually (3rd thoracic segment replaced by another copy of the 2nd).
Ed Lewis said a mutation happened and he thought that the 2 halteres has been replaced by 2 legs, but it had actually been transformed into a 2nd segment.
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Homeotic genes were first identified as dominant mutations that changed the identity of body parts e.g. Antennapedia in which legs develop in place of antennae.
There is a series of homeotic Hox genes in Drosophila that determine the identity of embryonic regions along the anterior-posterior axis. what do these genes encode for? do they occur individually or in clusters?
transcription factors with a conserved DNA binding domain (the ‘homeobox’ domain)
Hox genes occur in clusters, arranged in the same order as the regions they affect (‘colinearity’).
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Hox gene clusters are conserved between flies and mammals. Order of genes similar to flies as you can see- and same as order of expression along the anterior-posterior axis.
what's really cool about this though?
Some mammalian Hox genes are so similar to fly Hox genes that they can be swapped.
Hox genes probably played important roles in evolution - many differences in morphology between species/phyla appear to be due to evolutionary changes in Hox genes.
there are some differences here though: mouse just has more + on different chromosomes, some have been duplicated (crucial for evolution).
