GeneticsFinal Flashcards
(206 cards)
1
Q
How is gene expression regulated in prokaryotes?
A
Transcription initiation is one of the main mechanisms for regulating genes in prokaryotes.
2
Q
What is an operon?
A
It is a transcript unit coding for multiple proteins under a single promoter. Many prokaryotic metabolic genes are involved in the same process and are organized into operons.
3
Q
What is RNA polymerase?
A
It is enzyme that catalyzes the synthesis of RNA molecules from a DNA template during transcription. For transcription to become initiated: (a) RNA pol binding to promoter (b) initiation.
In prokaryotes, RNA polymerase easily binds to the promoter, default state of genes is “on”
4
Q
Where does the sigma factor of subunit of RNA pol bind?
A
It binds to the -35/-10 promoter sequences to properly position the holoenzyme at the transcription start site.
5
Q
What is the operator?
A
It is the binding site for repressors.
6
Q
What are genetic sensors?
A
Allosteric regulation of transcriptional activators/repressors.
7
Q
What affect does an ‘inducer’ have?
A
If it is there, its presence leads to increased gene expression, it will turn the gene on.
8
Q
If there is no lactose present in the lac operon, what is the outcome?
A
Does not want to express genes, repressor binds to operator, prevents RNA pol to transcribe genes, no mRNA in this state.
9
Q
If there is lactose present in the lac operon, what is the outcome?
A
Lactose acts as effector in the protein, binds repressor proteins, cause allosteric shift, now RNA pol is free and can move along the DNA, make mRNA copy, and now messenger RNA.
10
Q
What is ‘partial diploid’?
A
It is a mutation that results in duplication of a segment of its DNA, resulting in two copies of some of its genes.
11
Q
What are three main classes of lac mutants?
A
1) Structural gene mutations: affect function of just one enzyme - other is inducible.
2) Uninducible mutants: can’t make Lax Z and LazY in the presence of inducer (IPTG)
3) Constitutive mutant: make both LacZ and LacY, even in absense of Inducer.
12
Q
What are partial diploids used for?
A
Allows to test for dominance/recessiveness and critical in determining ‘cis’ vs ‘trans’ acting factors.
13
Q
What is the difference between cis and trans?
A
- Cis only effects the transcription of genes on the same DNA molecule
- Trans only effects transcription of genes on other DNA molecules
14
Q
Are promoters and operators cis-acting or trans acting?
A
They are cis-acting.
15
Q
Describe Lac operon regulation by P mutations
A
P- affects inducibility of both B-galactosidase and Permease - failure to “turn on”
Only effects transcription of genes physically attached to it on the same DNA molecule.
ie. chromosome or plasmid because the promoter is a cis-acting element.
Example:
P+Z+Y+/F’P-Z’Y- = Inducible
P-Z+Y+/F’P+Z-Y-= Uninducible
16
Q
Describe Lac operon regulation by O mutations
A
- Oc = O- is constitutive, both B-galactosidase and Permease are constitutively active, failure to keep “off”
- similar to P, only effects transcription of genes physically attached on the same DNA molecule, operator is acting in cis-acting element
Example:
O+Z+Y+ Inducible
OcZ+Y+ Constitutive
O+Z-Y-/F-OcZ+Y+ Constitutive
O+Z+Y+F-OcZ-Y- Inducible
17
Q
Describe lac operon regulation by I- mutations
A
- I- is similar to O, since both B-galactosidase and Permease and constitutively active BUT I does not have to be on the same DNA molecule, it can act in trans.
- I+, Z+, Y+ = inducible
- I-Z+Y+= constitutive
- I+Z-Y-/F-I-Z+Z- Inducible
18
Q
Describe lac operon regulation: Is mutation
A
- it is a super repressor
- it is a different form of I-, it is dominant to I+
- acts in trans
- always cause it to be inducible
- Is mutations affect the allosteric site of LacI
19
Q
How do glucose levels regulate cAMP levels?
A
- If there is high glucose, then ATP does not make cAMP.
- If there is low levels of glucose, then ATP does make cAMP.
20
Q
How does cAMP-CAP complex activate transcription?
A
- CAP binds to cAMP molecules and creates CAP-cAMP complex.
- Then the complex binds to Promoter just upstream of RNApol. This increases transcription.
21
Q
What are the conditions if bacteria wants to use glucose, but does not want to make lac operon genes?
A
- There is glucose present
- cAMP levels are low
- no lactose
- no lac mRNA
22
Q
What happens to a bacteria that has glucose, wants to use it, but then you add lactose?
A
- The bacteria utilizes the glucose and it represses the lactose even though it is present.
- levels of cAMP are low.
- very very little lac mRNA.
23
Q
What happens to the bacteria if there is no glucose, but only lactose?
A
- There is no glucose present
- cAMP levels are high
- lactose is present
- transcription is activated by CAP-cAMP
- there is a abundant amount of lac mRNA present
24
Q
What is the difference between bacterial & eukaryotic transcription?
A
- still cis acting DNA sequences and trans acting proteins
- but there is a lot more of everything
Bacteria: activator protein on RNA pol = on, repressor protein= off
eukaryotic: RNA pol II = on
25
What form of chromatin do transcription factors and other molecular processes favour?
They favour euchromatin, the less packed and more spread it is, the better.
26
What are examples of cis-acting DNA sequences?
- core promoter
- promoter-proximal region
- enhancers/silencers
27
What are examples of trans-acting proteins?
- general transcription factors
- common transcription factors
- cell/tissue specific transcription factors
-transcription cofactors
28
What does efficient transcription of genes require?
Binding to enhancer sequences.
29
What is the difference between enhancers and silencers?
Enhancers promote transcription, and silencers prevent it.
30
What do transcription cofactors lack that transcription factors have?
They lack DNA binding domain
31
What is the PAX6 gene?
- it is a transcription factor
- involved in eye development in mammals
- different enhancers regulate PAX6 expression in specific tissues
- downstream of intron
- only applies to retina
32
Describe the Yeast GAL system
- molecule formula of glucose and galactose are identical
- difference between them is orientation of hydroxyl group @ 4th carbon
- glucose is the preferred monosaccharide sugar used in metabolism
- yeast can concert galactose to glucose-1-P for energy and carbon metabolism.
33
What are the four galactose-responsive enzymes required for the process?
GAL 1,-2,-7,-10
34
What are the three regulatory proteins required for the process?
GAL3, -4,-80
35
What is the function of GAL4?
- It is a key pathway, transcriptional activator that binds to UAS
- Binds to enhancers called "upstream activator sequences" located upstream of GAL enzymes
- each GAL gene has its own promoter - not organized into an operon.
- UAS sites are located at a distance from promoters, not immediately adjacent.
- Activation domain of Gal4 helps recruit chromatin modyfying proteins, Gal4 activation domain can function in many eukaryotic cells, insects, humans, etc.
36
Describe the mutational analysis of GAL4:
GAL4-: Gal enzymes uninducible
GAL80-: GAL enzymes constitutive
GAL3-: Gal enzymes uninducible
- Gal80 binds to Gal4 activation domain and blocks activation.
- binding of galactose and ATP changes structures of Gal3, causes Gal3 to bind to Gal80, remove from Gal4 (i.e. Gal3 acts as a sensor and inducer and makes it active again!)
37
Enhancer-bolcking insulators restrict the action of enhancers to specific genes. What is an example of one?
- CTCF
- leads to chromosomal DNA into sub-domains/loops (TADs) which are essential for proper regulator.
38
Chromatin often needs to be remodelled locally to allow gene expression. This is to make binding sites accessible which are not due to incorporation in nucleosomes. What is an example of a nucleosome remodelling complex?
- Swi/Snf uses energy from ATP to reposition or remove single nucleosomes, exposing binding sites.
- It is a coactivator, it does not bind to DNA itself, it gets recruited by enhancer-bound transcription factors.
39
In terms of "histone code", define WRITERS, ERASERS, and READERs.
- Histone modifications are added by histone code WRITERS
- Histone modifications are added by histone code ERASERS
- Histone modifications are recognized and bound by histone code READERS
- readers are often coupled with writers or erasers. Different readers recognize different elements of histone code.
40
What is the effect of Histone acetylation on chromatin and therefore transcription? What would give the opposite effect? How would this work?
HAT (writers) would turn condensed chromatin (heterochromatin) to open chromatin (euchromatin), and HDAC (erasers) would do the opposite.
- Histone tail acetylation on lysines neutralize positive charges, loosens interactions with DNA (negative charge) and chromatin relaxes. Therefore there is increased accessibility and transcription. It creates binding sites for histone code readers that promoter activation.
41
What is the result of Histone tails being methylated? What enzyme is responsible for this?
- this will happen at lysine or arginine residues
- catalyzed by histone methyltransferase (HMTase) enzymes
- does not change the charge of the histone
- many histone methylation marks (H3K9Me) are associated with gene silencing, and act as a signal to recruit specific readers.
- promotes heterochromatin formation and tends to spread on chromatin.
42
What is the effect of HP-1 (Heterochromatin Protein 1)?
- It binds methylated histones, H3K9Me
- Promotes heterochromatin formation, recruits additional HMTase
- HMTase methylates neighbouring nucleosomes (chain reaction)
43
How would you stop the spread of heteorochromatin?
- Barrier insulators
- it is counteracted by HATS bound to barrier insulations which are DNA sequences
44
What are epigenetics?
The study of heritable traits that cannot be explained by changes in DNA sequence. Many modifications to DNA and chromatin structure are inherited.
45
Explain the epigenetic phenomenon in Drosophila eye:
- Brown + red pigments contribute to Drosophila eye colour
- "white" (w) gene codes an ATP-binding cassete (ABC) transporter that carries the precursors of red and brown pigments in developing eyes
- Saw WT and wild mutants.
46
What is Position-effect variegation? And what is an example? How did they discover this?
Position-effect variegation (PEV) is a phenomenon observed in genetics where the expression of a gene or genes is affected by their position in the genome
- Epigentic silencing by heterochromatin spreading is an example
- They mutagenized flies with X-rays, screened for offspring with unsual phenotypes and looking into neighbouring regions and which lead to the silencing.
47
How did they identify proteins that promoted or prevented the spread of heterochromatin? What were the results?
- They did screening, second-site mutations that effected spreading of heterochromatin and could see what made it better or worse.
Results:
- HAT (histone acetyltransferase): spreading enhanced, more white+ silenced.
- Histone Methyltransferase and HP1: Spreading were supressed, fewer white+ were silenced.
48
What are the two principles of Mendel's Law?
1) Each individual has two allele for each gene, passes only one to offspring at random.
2) Each allele has an equal chance of being inherited (independent assortment)
49
Describe Non-mendelian genetics
Pattern of inheritance in which traits (phenotypes) do not seggregate in accordance with Mendel's Laws. The phenotype cannot be explained like this.
50
What is dosage compensation by X-chromosome inactivation?
- Dosage compensation is done so females and males have equal expression levels (around 1000 genes) located on the X chromosome. (around 80% get silenced by X inactivation)
- This explained the Non-mendelian inheritance of coat colours in toitoiseshell cats due to X-chromosome inactivation
- There was black and orange fur
- They saw that there were X-linked diseases that only applied to males and not females, for example males are colour blind, but if there was a skewed X inactivation for females, they could be colour blind as well.
- The inactive X in humans is silenced by repressive histone and DNA marks
- Dosage compensation by X-chromosome inactivation is a process that occurs in female mammals to equalize the expression of genes on the X chromosome between males and females. Since males have only one X chromosome, while females have two X chromosomes, if both X chromosomes were to remain active, females would produce twice as much of the proteins encoded by X-linked genes as males. To avoid this imbalance, one of the X chromosomes in each cell of a female mammal is randomly inactivated during embryonic development.
51
DNA methylation
- catalyzed by DNMT (DNA methyltransferase enzymes)
- occurs primary on cytosine in CpG dinucleotide
- 60-80% of CpG are methylated genome-wide in vertebrate.
- CpG methylation is not randomly distributed, mostly associated with intergenic regions, heterochromatin.
- associated with decreased levels of transcription.
- CpG island are clusters near promoters.
52
How can methylation associated with repression/silencing of gene expression be direct and indirect?
Direct effect: DNA methylation blocks transcription factor binding
Indirect: Due to recruitment of HDACs and HMTs that lead to repressive histone modifications.
- all can be passed through mitosis- heritable
- DNMTs have high affinity for hemimethylated sites. (guided by methylation pattern on parental strand)
53
What is the unusual pattern of inheritance of the Igf2 gene involved in embryonic growth?
- "Insulin-like growth factor 2"
- Binds to receptor on cell surface, stimulates cell to grow.
- Autosomal gene.
- Only if mutant allele inherited from father.
54
What is genomic imprinting? What is monoallelic inheritance?
- imprinting = silencing
- up to around 200 genes in humans and mouse, genomes only paternal or maternal, not both, expressed - as is there was only one copy of gene in the cell
- monoallelic inheritance
- sex-specific gene silencing
- non-expressed allele is said to be Imprinted
- imprinted copy is inactivated by mechanism involved in DNA methylation in maternal or paternal germ line
- maintained throughout life of progeny in somatic cells
55
In mice and humans, how are Igf2 and H19 genes imprinted?
- They are adjacent
- Igf2 is maternally imprinted, only inherited from father is expressed. maternal copy is silenced
- opposite is true for H19 gene, paternal is imprinted and mother is expressed.
56
What is an alternative way of methylation at imprinting control region determining gene expression?
- Sex-specific CpG methylation of imprinting control region, ICR - only seen in paternal gametes (sperm).
- Unmethylated ICR binds to to CTCF, acts as an enhancer-blocking insulator, so if it was methylated, it would prevent this, and H19 promoter would silence transcription.
57
How would dwafirsm happen?
- Faulty imprinting
- effected individual did not methylate/silence the paternal allele. So Igf2 is expression is repressed which is should not be. and then this is the result.
58
As a result of offsprings inheriting many things: proteins, mRNAs, and other RNAs, organelles etc. What are some things we could study?
Cytoplasmic inheritance, maternal inheritance, extra-nuclear inheritance.
59
What are 3 factors influencing the replication of mitochondrial genome?
1) Mitochondrial DNA is replicated within nucleoids
2) Nucleoids can divide within an organelle.
3) Mitochondria themselves divide.
60
Why is mitochondria inherited from the mother?
- maternal inheritance - cytoplasmic inheritance
- way more mitochondria in oocyte then sperm, upon fertilization, the few sperm in the mitochondria are destroyed (or consumed) by the oocyte.
61
What can be used as a tool to trace maternal heritage?
mtDNA
- can lead to heteroplasmy
- higher rate of mutation than nuclear genes, more frequent DNA replication, no DNA repair.
- spont. mtDNA mutations can lead to two distinct mt populations within a single cell - heteroplasmy.
62
What is an example of cytoplasmic inheritance (neurospora)?
Brown= not normally functional mitochondria
Green = normal
- if offspring is mutant poky, 100% affected/mutated is a result of mother
- maternally inherited
63
What does homoplasmic mean?
Random segregation of organelles at mitosis (or meiosis) can lead to cells that are homoplasmic, which means they are identical.
64
What are the traits of human diseases caused by dysfunction in mitochondria?
- effects at least 1/5000 people
- progressive
- multi-system disease (high-energy demand tissues)
- some autosomal, some inherited different ways, depends.
65
Describe the pedigree of human mitochondrial disease caused by mitochondrial genes? What is the solution?
- children of affected mother all have it
- children of affected farther do not have it
- in some cases if mother is heteroplasmic (mix of normal and non-normal) children may be normal.
- Solution: pronuclear transfer - three person in vitro fertilization: resulting embryo has 3 parents.
66
Describe the embryonic development in Drosophilia over 24 hours.
(a) sperm and egg nuclei fuse to create single-celled diploid zygote
(b) multiple nuclear divisions create a single multi-nucleated cells. (70 mins)
(c) nuclei migrate to outer embryo and divide several more times, creating syncitial blastoderm. (120 mins)
(d) cell membrane grows around each nucleus, producing layer of cells that surrounds the embryo. Resulting in cellular blastoderm. (180 mins)
(e) nuclei at one end of blastoderm develops into pole cells, which become germ cells.
67
Define "cellurization"
When individual nuclei are enclosed in plasma membrane to form cells.
68
ZGA
transcription at embryos own genes begin
69
After 2 hours, what is defined in embryo?
Head, tail and back/front regions.
70
After 10 hours, what happens in the embryo?
body plan gets subdivided into "segments"
Each segment goes on to form specific structures
71
Describe the two screens performed to identify genes required for organizing the Drosophila embryo.
Two screens: Maternal vs Zygotic genes
Screen 1: What mutations in the mother prevent her offspring from complementing embryonic development?
-cytoplasmic inheritance
- maternal-effect genes - phenotype determined by genotype of MOTHER.
Screen 2: which of the embryo's own genes are needed for it to develop normally?
- zygotic genes - phenotype determined by genotype of the embryo
- inheritance shows "normal" mandelian pattern.
ex. mim homozygous - mutant phenotype.
72
Describe the cascade of transcription factor expression tat regulates early Drosophila development.
Based on similarities of mutant embryo phenotypes, able to organize genes into 5 groups that act one after another.
73
What is the function of the two maternal-effect genes? What are they?
Anterior (A): bicoid (mutants lack this structure)
-bcd, encodes TF
- protein forms A [high] to P [low] concentration gradient
- it is sufficient - inject bicoid mRNA direct formation of anterior structure
Posterior (P): nanos (mutants lack this structure)
- [A] low to P [high]
- not a transcription factor, but a transcription regulator
- Nanos inhibits transcription of uniformly-distributed maternal hunchback mRNA.
74
What are the function of Gap genes?
They translate maternal A-P gradients into broad subdomains.
- 9 gap genes identified
- (eg.) zygotic hunchback (hb-z),knuppel, knips, giant.
- all encode transcription factors.
- transcriptionally regulated by maternal-effect gene products.
- they are characterized by loss of several consecutive segments, corresponding to the to the region in the embryo where gap genes was transcribed - large gaps in their body plan.
- transcriptionally regulated by maternal effect gene products.
(there are 3 binding sites for Bcd in Hb promoter)
75
What are pair-rule genes?
-Further subdivision of the embryo
- pair-rule gene mutants are characterized by the absense of every other segment (even skipped - odd skipped)
- 8 identified
- all transcription factors
- expressed in 7 stripes, position of stripes depending on gene
- helps define 14 segments of embryo
- regulated by maternal effect and gap gene
- even skipped gene (eve), nuclei in stripe 2 will have specific concentrations maternally loaded and zygotic factors.
- High [Hb-z]
- Low [Bcd}
- Low [Gt],[Kr]
76
What are stripe-specific eve enhancers?
- eve: even skipped
- each enhancer has diff. arrangements of binding sites for maternally loaded factors and Gap genes.
- This allows enhancers to activate eve transcription in a specific stripe of nuclei.
"combinational control of transcription"
77
Describe the establishment of segmentation domains:
- Segment polarity genes
- encode components of two cell-cell signalling pathways, includes secreted proteins, membrane receptors, transcription factors...
- activated/repressed by pair-rule genes
- function t define A and P within each segment have mirroring of one half of each segment
78
What are homeotic mutations?
- mutant animals lack a particular structure, which is replaced by another structure, normally fond in other body segments (homeotic transformation)
- ex) (Ubx) - second thorax and set of wings in place of halteres
- Antp - leg instead of antenna
79
What are hox genes?
- found in the establishment of segment identify
- 8 genes, all encode homeodomain family transcription factors
- expressed in specific segments, some overlap
- activated/repressed by Gap and pair-rule gene products
- diff structures depending on Hox genes.
80
What is the physical basis of the mendelian law of heredity?
- chromosomes occur in matched pairs
- chromosome pairs segregate 1:1 in meiosis.
- chromosome pairs segregate independently.
- segregation patterns correlate w/ inheritance of traits.
81
What affect would 3n have?
Both banana and strawberry are 3n, no seeds, if there is not 3n then you have seeds.
82
Define euploid
good, two sets, nothing extra and nothing missing on a chromosome.
83
Define aneuploid. How does it arise?
abnormal amount of chromosomes
Ex. 2n+1, 3 at chromosome 21.
- through abnormal segregation of homologous chromosomes: due to unpaired chromosomes during meiosis in triploid (frequent), due to "non-disjunction" of homologous chromosomes during meiosis in diploid individuals (rare), failure to seperate.
- can happen at meiosis I or II, can be diff proportions and diff outcomes. 100% aneuploid at meiosis I , 50% anueploid at meiosis II.
84
Polyploidy
General term for having more than two sets of chromosomes. Uncommon in animals, but common in plants.
85
What are consequences of autoploidy?
Somatic: non-germ line cells, human: lethality
Germline: plants sterile, seeds do not develop, seedless (watermelon).
86
Why are triploids sterile?
Extra set of chromosome causes big problems during chromosome segregation in meiosis.
- triploids rarely produce euploid gametes
Probability P(euploid gamete)= (1)(1/2)(1/2)=1/4
87
What is the probability of watermelon n=11 of p(euploid)?
(1/2)^(n-1)
(1/2)^10
= 1/1024
88
Does meiosis happen in tetrapoids?
Yes, even number, chromosome can pair, 2n gametes. Euploid.
89
What causes autoploidy (within same species)?
1) Spontaneous
- fertilization by multiple sperm (eg. dispermy)
- error in meiosis leading to diploid germ cells.
2) Induced
- disruption of chromosome segregation (plants)
- polyploidy can be generated through selective breending or induced. (Colchinine)
- triploidy can originate through effective meiosis, also through dispermy, egg cell is fertilized by more than one cell. (new giant triploid invasive crayfish)
90
What is the difference between autopollyploid and allopolyploid?
Autopolyploid:
- individual that has multiple chromosome sets from within one species
Allopolyploid:
- Individual that has multiple chromosome sets originating from two or more diff. species
91
What is an example of allopolyploid?
ex)
Radish
n=9
2n=18
x
Cabbage
2n=18
n+n= 9 + 9
Sterile F1 hybrid
2n + 2n = 36
Fertile amphidiploid, one plant ends up fertile, chromosome number spontaneously doubled. Plant is also bigger, cross it back to one of the parent plants.
92
What is Trisomy and what is an example?
- it is viable aneuploidy in humans
- trisomy 21= down syndrome
- XXY = klinefelter syndrome
- XO: Turner syndrome.
- trisomy 13,18: non viable, usually dies at Infancy, all others if they are non viable, die in utero.
93
What is more tolerable to chromosome changes, plants or animals?
Plants
94
What is Cri-du-chat syndrome?
- different ways can rearrange
- tip of chromosome 5 is missing
1) genes in deleted region are important
2) genes are haploinsufficient
Where do these deletions come from?
- can be small or large
- breakage and rejoining
- crossing over between repetitive DNA
95
What is an example of a chromosomal deletion?
- Willians syndrom deletion
- associated with deletion of 17 genes on chromosome 7
- Found at frequency 1 in 10,000 people
- pronounced musical or singing ability, hyper-sociality
- caused by 1.5-Mb deletion on one homolog of chromosome 7, specifically at band 7q11.23
- use complementation test to test for deletions in chromosomes
96
How would you map a deletion? And how would you map a recessive mutant allele?
What is an example of this?
- known recessive mutant alleles can be used to map a deletion
- know deletions, can be used to map a recessive mutant allele
Example:
- fruit flies homozygous for this chromosome, have no eyes. The hypothesis is that they contain a mutation in gene that is important for eye development.
Example:
- fruit flies heterozygous for this chromosome have normal eyes, where is the "eyeless" gene located?
97
What are chromosomal rearrangements: Inversions?
Normal: AB C D E F
Paracentric: AB C E D F (does not include centromere)
Paricentric: ADC B E F (includes centromere)
98
What are the consequences of Inversion? Somatic vs Germ Line?
Somatic:
- within gene, C-C is disrupted.
- Gene A and D fusion.
- or it can be no disruption.
Germ line:
- recombination during meiosis I.
- Inversion loop --> segmentation. To get proper alignment.
Paracentric:
- 1 product would be normal, one would be inversion, and 2 would be broken with major deletions.
Pericentric:
- all products the same with major deletions.
99
What is the relationship between Inversion and chance of recombination?
The larger the inversion, the greater chance of recombination.
100
If you do not get the expected recombinant frequency, what could this indicate?
It could indicate fertility issues, you would then need to figure out what inversion took place.
101
What are the somatic consequences of balanced translocations? What is an example?
if there is an even swap, there is no net gain or loss of DNA content, then the phenotype would be normal. Few or no somatic symptoms. Lead to 50% defective gametes not being viable.
102
What is
Robertsonian translocation?
Robertsonian translocation is a type of chromosomal translocation that occurs when two acrocentric chromosomes (chromosomes with the centromere near one end) fuse at their centromeres, resulting in a single, larger chromosome
103
Describe grape-colour genetics:
- Wild type allele: carbernet (purple)
- loss of function allele: chardonnay (green) does not make pigment. has LTR retro-element. Transposable element.
- Intermediate strain: partially functional allele, has tiny bit of fragment LTR, event is likely mispairing/crossing over - between homogous sequences, could be a deletion.
104
How were transposable elements discovered?
- through the study of "unstable" alleles
- used maize as a system for studying the inheritance of traits (pigment genes)
- looking at the gene that gives the corn kernels its pigment, called Gene C (purple pigment)
- c is recessive allele that is unable to make pigment.
- C1 is dominant inhibitor that represses pigment production.
- normal 3:1 phenotype ratio in F2.
- when they crossed them and saw that they did not get all colourless kernels when crossing with C1, they released that there must be transposable elements. If the pigment can be made it must be that C1 is not working anymore.
105
Describe the discovery of Ds and Ac.
- the breakage of Ds requires a second genetic element called Ac
- Ds is stable without Ac
- With Ac, Ds breaks the chromosome causing blue sectors by uncovering C.
- Ds can also jump to a new location
- non-autonomous (like Ds) or autonomous (like Ac)
- Ac element can always jump but Ds can only with Ac.
- jump out early = large spot
- jump out late, can divide more so smaller spot.
106
What are examples of DNA transposons?
1) Ds and Ac elements in Maize
2) P-elements in Drosophilia
These are in prokaryotes and eukaryotes.
107
What is an example of a retrotransposon?
- similar to retroviruses
1) LINE - found in human genome
- MoMLV - two repeats each at one end, three genes in the middle.
- retrotransposons jump from one location to another via an RNA intermediate
- transposition is mediated by reverse transcriptase (encoded by retrotransposon pol gene) not transposase.
- copy and paste mechanism
108
Transposable elements are abundant in large genomes. What is the breakdown?
- 40% of the genome is transposable elements
- LINEs - class I - 21% autonomous
- SINEs - class I - 13% nonautonomous
- 3% class 2, DNA transposons
109
What is the relationship between plant size and the amount of transposons?
The bigger the plant, the more transposons.
110
Where are transposable elements usually found?
- Introns or intergenic regions. This is because if it goes into a gene, the insertion will be deleterious (cause harm), not be viable, and be selected against.
111
1/600 spontaneous mutations causing important human diseases result from transposition of a LINE or SINE element. What are 2 examples?
1) Homo-sapeins-specific LINE-1 (L1Hs)
- Insertion in a patient with homophilia A (blood clotting disorder F8) interupts a coding exon of vactor VIII (F8)
- insertion into an exon disrupts genetic code, now they have a disorder.
2) Insertion of AluYa5 element in patient with Dent disease interupts an exonic splice enhance (ESE). Skips exon 11, and introduces stop codon in exon 12. Disrupts splicing, 10 goes to 12, causes frame shift and non function protein leads to disease.
many cancers also associated.
112
What is genomic surveillance?
Limiting the impact of transposable element mobility. Cells have mechanisms to repress transposable element mobility and protect genome integrity.
113
How do cells repress transposable elements?
- did observations in C. elegans
- observation: Tc1 elements transpose in somatic cells but not germline cells.
- hypothesis: the germ line cells contain some kind of "silencing machinery" that represses Tc1 mobility. The components of this machinery are encoded by genes.
- to identify these genes, did "genetic screen" to identify which genes allowed Tc1 to mobility. Then infer which are knocked out, those have to do with silencing.
- If we insert Tc1 in unc-22 gene, then twitching/uncoordinated worm. If Tc1 mobilized, then it is stable, smooth gliding worm.
- identified 25 genes that blocked the mobility of Tc1. Many of the genes were involved in the Rnai silencing pathway.
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What are the steps of RNAi silencing pathway?
- many steps, including cleavage of dicer
- once fully processed, mRNAs bind to RISC complex
- miRNA/RISC complex recognized mRNA targets through sequence complementairy and either promotes their degredation or represses their translation.
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What are PiRNAs?
Repress transposon mobility in germ line of Drosophilia and other animals.
Present in P strain, not in M strain.
Repress mobility to normal.
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What is the function of p53 protein?
- it silences transposable element mobility
- there is a mutated/absent p53, there is destabilized genomes, cancer, inflammation, sporadic diseases.
- mutation found in most tumour types.
- p53 protein is required for silencing transposon mobility in Drosophilia
- human p53 corrects dysregulated transposon activity in p53-flies, but p53 variants commonly see in cancer patients do not.
- p53 keeps retrotransposons silent
- p53 promotes genome stability by silencing transposon mobility
Example:
Wilms (kidney) tumour
- loss of p53 in kidney cells leads to deregulation of retrotransposon activity and to tumourigenesis.
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Chromosomal mutations
Gain or loss of all or part of a chromosome
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Insertional mutations
Insertion of large regions of DNA, (transposable elements)
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Point mutations
Changes in a single nucleotide or addition/deletion of one or more nucleotide.
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What are the two categories of point mutations?
1) Transition
GC -> AT
2) Transversion
CG -> TA
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Synonymous mutations (silent mutations)
Change sequence but not the amino acid it encodes
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Nonsynonymous mutations (missense mutation)
- change the sequence of a codon to one that codes for a different amino acid
1) conservative mutation: mutant codon encodes a chemically different amino acid (eg. lys to arg)
2) non-conservative: mutant codon encodes a chemically different amino acid (e.g. lys - to -thr)
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Nonsense mutation
- change the sequence of a codon that codes for an amino acid into one that stops translation (ie. UAA, UAG, UGA)
- effects depends on distance from 3' end of ORF.
- can trigger nonsense-mediated decay (NMD), which completely degrades the mRNA..
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Frameshift mutation
- caused by insertion/deletion
- change the translation reading frame for all codons downstream of the mutation
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Define hypomorphic
weak/partial protein retains some function or is produced at a reduced level.
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Null
protein is non-functional or not produced.
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hypermorphic
protein is hyperactive
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ectopic
more protein made or made in wrong time/place
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Neomorphic
protein gains new function
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What can mutations in non-coding regions disrupt? What is an example?
Can disrupt transcription, splicing, stability, translation, etc.
Example: BAP1 synonymous mutation results exon skipping, loss of function and worse patient prognosis.
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What can be an effect of early stop codon?
It can lead to mRNA degradation via nonsense mediated decay. Which can lead to no RNA and protein being produced at all.
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What is the effect of a point mutation on a gene's mRNA and protein products?
- could lead to detectable changes in mRNA or protein generated from gene
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What is the difference between northern blots and western blots?
Northern blots are to look at RNA and western blots are to look at protein.
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What is the difference between an incorporated error and a replicated error?
Incorporated error: can be repaired.
Replicated error: permanent.
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What is the origin of spontaneous mutations?
1) Errors in DNA replication
- base mispairing
- strand slippage
strand slippage during DNA replication, leads to Indels, whether slippage generates insertion or deletion, that depends on which strand loops out first.
Greater N = greater slippage
2) chemical changes to DNA (leads to errors in DNA replication)
- depunnation
-deamination
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What do trinucleotide repeat disorders arise from?
They arise from expansion of repeated sequences (slippage during replication)
- diseases caused by expansion of non-coding trinucleotide repeats.
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What is the function of FMR1?
- it is important for normal brain development
- methylation of trinucleotide repeats blocks transcription of FMR1.
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What is an example of permanent spontaneous chemical change to DNA?
- Oxidative damage
- potentially mutagenic, blocked DNA replication and transcription.
- G C --> T A Transversion.
- caused by certain products generated in the mitochondria by normal aerobic metabolism of molecule oxygen.
ROS = reactive oxygen species include molecules like superoxide radicals etc.
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What are two permanent mutations and what is the logic behind it?
Permanent mutation: deamination and depurination.
Logic: addition of incorrect base during DNA replication can lead to permanent point mutation.
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What are some examples of induced mutations?
- UV light
- ionizing radiation
- exogenous chemical agents
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What is the Ames test? What is the problem with it? And what was the improvement that was done?
Ames test: If treatment of these his-mutant salmonella cells with a particular compound increases the number of revertants, then compound is mutagen.
- use different starting strains to test for different types of mutations.
Problem:
- bacteria is not people
- add compound to bacteria, humans have digestive system, liver, bacteria, need to look at metabolites not just chemicals.
Improvement:
- revised ames test:
- add liver enzymes to test chemicals that might become mutagenic only when metabolized.
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What is the difference between forward genetics and reverse genetics?
Forward genetics is an approach in which researchers start with a particular phenotype (observable characteristic or trait) and try to identify the gene or genes responsible for that phenotype. This is typically done through the use of mutagenesis, in which random mutations are induced in a population of organisms and individuals with the desired phenotype are selected and analyzed to identify the mutated gene. This approach is useful for discovering new genes and understanding the functions of known genes.
Reverse genetics, on the other hand, is an approach in which researchers start with a known gene and try to determine its function by studying its effects on the phenotype. This is typically done through techniques such as gene knockout, in which the function of a particular gene is disrupted or eliminated in an organism to observe the resulting changes in phenotype. This approach is useful for understanding the specific functions of known genes and their role in biological pathways and processes.
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How do we understand all the sequence information, what do we look for?
1) look for protein encoding information
- exons
- introns
2) open reading frames as evidence for possible exons. Run it, if it does not stop with a stop codon then it is an ORF.
3) Check for "codon bias" as evidence that an ORF is a part of a real gene.
- look for the codon preference as amino acids can be coded by more than one codon, this relfects the relative tRNA abundance and can be signature of an oRF that is part of a real gene.
- check for conservation of a predicted ORF across species as evidence that it is part of a real gene. (higher bar --> seen in evolution)
3) look for cDNAs that map to the putative gene (best evidence) - look if the gene is expressed or not.
- conservations and cDNAs help identify a 'real' gene
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What is an EST?
Expressed sequence tag - is a cDNA that has not been completely sequenced
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Comparison between organisms
to compare types of genes and their arrangements in the genome and to infer information about genome structure and evolutionary processes.
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Comparison within an organism
to identify gene families and gene duplication
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comparison between individuals of the same species
to identify differences associated with phenotypes or disease.
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When would you perform exome sequencing?
In human medicine. Just sequencing the "exome" can be effective alternative to whole genome sequencing.
Many mutations associated with disease affect coding regions of genes.
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Describe the "dog project"
There was a locus linked to sociability in dogs and lacking in wolves. Corresponds to the human chromosomal deletion associated with william syndrome in humans, which is associated with hyper sociability.
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Comparing genomes between species: Phylogenetic inference
relationships between homologs, orthologs, and paralogs.
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orthologs
homologous genes at the same genetic locus in different species, evolutionary inhereted from a common ancestor.
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paralogs
homologous genes at different loci at the same species, having arisen from gene duplication.
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Synteny
The conserved order of genes between two genomes is called synteny.
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Explain how comparing genomes can reveal "parent-child relationships"
- found out: child exhibiting uniparental disomy, means they have two copies of a chromosome from one parent and no copies of that chromosome from the other parent. happens from non-disjunction.
- UDP is generated by non-disjunction in meiosis, followed by "trisomy rescue" in the embryo.
- not all chromosomes are equally likely to be found in UPD.
1) Heterodisomy (HetUPD)
- both homologs from one parent are represented in the child.
2) Isodisomy (IsoUPD)
- only one homolog from one parent is represented in the child.
3) partial isodisomly (partial IsoUPD)
- if crossing over occured during meiosis, then disomy will be a mix.
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Antigen
any molecule (usually a protein) that elicits an immune response.
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Antibody
(also called immunoglobulins) proteins (present in the blood and other body fluids) that bind to antigens, marking them for destruction by phagocytic cells.
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Autoimmune diseases
an immune reaction against its own antigens (proteins)
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Humoral immunity
production and secretion of antibodies by a specialized lymphocytes (white cells) called B cells.
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Cellular immunity
specialized lymphocytes called T cells produce T-cell receptors that recognize and bind antigens found only on the surface of the body's own cells.
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How do humoral (B-cell) and Cellular (T-cell) immunity develop?
- each B cell and T cell recognize a unique non-self antigen that elicit immune response (diversity in antibody and T cell receptor)
1) B cell:
- lymphocytes originate from stem cells in the bone marrow
- B cells mature in the bone marrow
- when B cells encounter antigens, they mature into plasma cells, which secrete antibodies that bind to the antigen.
2) T cell
- T cells mature in the thymus and enter circulation.
- they attack by binding host cells and lysing them
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Describe the clonal selection of B-cell that binds to a specific antigen. followed by the memory B cells that have been activated.
1) In a large pool of B lymphocytes, each is specific for one antigen.
2) When an antigen binds to a B cell, the B cell divides.
3) gives rise to a clone of B cells, all specific for the same antigen.
4) this proliferation of lymphocytes is the primary immune response.
5) some cells differentiate into antibody-secreting plasma cells.
6) Antibodies are specific for the antigen.
7) memory cells remain in circulation.
8) if a second exposure of the same antigen occurs, the antigen binds to the memory cells. Which rapidly gives rise to a secondary immune response.
- similar process occurs for T cell and T cell receptor
- fast immune response when exposed to mutagen a second time
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What is the reason why vaccination works?
- The second immune response
- The active agent (the antigen) of a vaccine:
- Intact but inactivated (non-infective) pathogen
- Attenuated (reduced infectivity) forms of the pathogen
- purified components of the pathogen that have been found to be highly immunogenic
- the vaccine thus induces the primary immune response, generating the memory cells that are ready when the (second) infection happens.
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What are immunoglobulins?
The proteins that protect us.
Two identical light chains and two identical heavy chains.
- B cells produce a unique light and heavy chain combination that can recognize a specific antigen
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What are three ways of generating diversity?
1) Somatic recombination
2) Junctional diversity
3) Somatic hypermutation
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What is the function of RAG1, RAG2, and DNA repair enzymes?
They introduce dsDNA breaks and joint random V and J segments. This occurs at the level of DNA.
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What is the different in segments between the light chain and the heavy chain?
- Pre-mRNA is spliced to produce unique combination of V-J-C light chain
- Heavy chains have D (Diversity) segment in addition to V J and C segments
- V, D and J segments contribute to the antibody specificity, the constant regions of the heavy chain do not.
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What can junctional diversity result in?
- It is an imprecise junction.
- few random nucleotides are lost or gained. In many cases, it will result in a frameshift that produces a nonfunctional gene.
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What is somatic hypermutation?
- The immunoglobulin genes are subject to a high mutation rate
- deamination of cytosine which become uracil
- uracil is replaced by the DNA repair mechanisms by another base, thus generation a point mutation.
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What is the diversity of a T-cell receptor?
- they are composed of alpha and beta chains that have variable regions
- alpha chain: 44-46 V gene segments, 50 segments and a single C segment
- beta chain is similar to alpha chain, but contains D gene segments.
- somatic recombination and junction diversity, but no hypermutation.
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What are the 6 hallmarks of cancer?
1) Resisting cell death
2) Inducing angiogenesis
3) Enabling replicative immortality
4) Sustaining proliferate signalling
5) Evading growth suppressors
6) Activating invasion and metastasis
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What genes are associated with hereditary breast cancer and ovarian cancer syndrome?
BRCA1 abd BRCA1
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What gene is associated with Li-Fraumeni syndrome?
P53
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What gene is associated with familial adenomatous polyposis?
APC
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What gene is associated with retinoblastoma?
RB1
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In humans, most proto-oncogenes are activated in the absense of viral infection: how?
- mutations in coding sequences
- chromosome abnormalities: increased expression, fusion protein (cancer specific form)
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What is the function of sos protein?
- stimulates GDP-GTP exchange
- it is a guanine nucleotide exchange factor
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What are chromosomal changes that are associated with certain types of cancers?
- Reciprocal translocation between Ch 8 and Ch 14 t(8:14), observed in Burkitt lymphoma (B-cell)
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What is the function of c-Myc?
Transcription factor whose normal function is to promote cellular growth/proliferation (proto-oncogene)
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What happens in cancer by translocation? what is an example?
- Relocation of an oncogene next to a novel regulatory element: Burkitt lymphoma
- the coding sequence of the oncogene is NOT changed
- the same protein is overexpressed
- also frequently observed in CML (Chronic Myelogenous Leukemia_
- philidelphia chromsome 9:22 observed in 95% of people with CML
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What is c-Abl?
Protein kinase is involved in many cellular processes (proto-oncogene)
- an join onto Bcr1-Abl and become a hyperactivated kinase and then it is a oncogene, fusion protein.
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What is the function of gleevec?
it is a cancer drug that was developed specifically to inhibit Bcr-Abl
With this treatment, the five-year survival rate for CML has nearly doubled.
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What does pedigree analysis show of retinoblastoma?
that 40% of the cases involved an inherited (familial) mutation.
- may also be unilateral (affecting only one eye) or bilateral (affecting both eyes)
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What do viral oncoproteins from DNA tumour virus bind and inactivate?
Rb and p53
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Describe the clonal evolution of cancer cells:
- A single cell acquiring a mutation that provides proliferative advantage gives rise to a clone of mutated cells
- A single cell from the clone may acquire a secondary mutation that provides additional proliferative advantage
- eventually, cycle of acquiring mutations and clonal expansion gives rise to fully transformed cancer cells
185
Describe cell hybridization/fusion:
- cell fusion generates hybrids containing genomes from the two different cell lines
- the genomes of fused cells are unstable, randomly keep or lose chromosomes
- in hybrids, between rodent and human cells, rodent chromosomes are predominantly kept and most human chromosomes are lost (this is used to map GOI in human)
- cell fusion is highly inefficient, a selectable marker is necessary to identify hybrids.
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What is the HAT medium?
Cells have way to synthesize nucleotides, to be able to use them to replicate DNA.
Can produce dntp through Novo
pathway.
Use this to make new DNA and replicate it.
Recycle is the Salvvage pathway.
Need HPRT for dGTP and TK for dTTP.
If you put into HAT medium (drug/poison) that will inhibit Novo pathway. and then they will die, can not make enough dntp. unless it is in presence of
aminopterin. HAT medium gets rid of one of the important enzymes.
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If you grew TK deficient cell and HPRT deficient cell in the HAT medium what would happen? (both important enzymes of the salvage pathway) What if you fused both lines together?
If you grew them in HAT medium then they would both be dead. But if you fused them together then you have a newly synthesized DNA, live.
188
What is gene therapy and why?
Gene therapy involves adding a normal (wild-type) copy of a gene to the genome of an individual carrying defective (mutated) copies of the gene, often recessive mutations.
Only a few of the 4000 inherited human diseases are currently treatable.
189
What are two viral vectors?
1) Derived from Adenovirus
- will infect all cells even non-dividing ones
- the vectors will not be integrated in the genome, thus the transgene is diluted and eventually lost.
2) Derived from Retrovirus
- many will infect only dividing cells, but others (HIV) can infect without host cell division.
- the transgene and viral vector will be incorporated into the genome of the infected cells (more stable)
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What is the difference in gene therapy in somatic cell vs germline?
somatic cell: gene therapy refers to the transfer of a gene in somatic cells
germline: gene therapy refers to the transfer of a gene in all cells of an organism through the germline transmission.
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What is ADA-SCID?
- rare autosomal disease of the immune system (bubble boy disease)
- affected individuals have essentially no immune system:
- in the absense of ADA, deoxyadenosine accumulates in T lymphocytes and eventually kills these cells. T lymphocytes are responsible for the stimulation of the anti-body producing B lymphocytes.
- most common treatment is bone marrow transplantation
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What are some issues with conventional gene therapy? what is a potential solution?
- Integration sites cannot be controlled
- Expression level of the rescue gene may not be optimal
- Ex vivo experiment is limited to certain types of cells
- potential solution: directly edit genomic sequences of stem cells from the patient.
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What are tools to target specific sequences in the genome?
- the clustered regularly interspaced short palindromic repeats (CRISPR) system uses a RNA molecule to target specific DNA sequences
- to target different sequences in the genome, one has to simply change the crRNA sequences.
194
What is blending theory?
Traits of parents get mixed, like fluid, in the offspring, resulting into new traits that resembles parents.
195
What is the reason why Mendel's experiments worked?
- traits affected by only one gene. Many traits (people's height) are affected by multiple genes.
- pure genetic background and ability to cross - or self- pollinate (ability to control cross/mating)
- ability to obtain a large number of progeny
196
Why is the ability to obtain a large number of progeny important?
- A large sample size reduces the variability between experiments
- A large sample size helps to determine precise data
- low probability events can happen at any time during the experiment
197
What cross does monohybrid cross make?
3:1 ratio
198
What is the chromosomes theory of inheritance?
It was that chromosomes, like mendel's "elements" come in matched (homologous) pairs in an organism.
The members of homologous pair seperate during meiosis, so each sperm or egg receives just one member.
The members of different chromosome pairs are sorted into gametes independently of one another in meiosis, just like the alleles of different genes in Mendel's.
199
What can pedigrees tell us about trait/phenotype/disease?
- dominant or recessive?
- autosomal or sex-linked?
- common or rare trait?
200
What are the 7 traits that Mendel used to demonstrate independent assortments?
1) Round or wrinkled ripe seeds
2) Yellow or green seed interiors
3) Purple or white petals
4) Inflated or pinched ripe pods
5) Green or yellow unriped pods
6) Axial or terminal flowers
7) Long or short stems
201
Chromosome segregation of a dihybrid during meiosis
Independent assortment, resulting in equal ratio of four different genotype.
202
What does dihybrid testcross produce?
- It produced equal number of parental and recombinant type
- the ratio of 50/50 between parental type and recombinant if the two genes are on different chromosomes "independent assortment"
203
What is the Chi-square test used for?
A way to determine the deviation of observed values from expected values is simply produced by chance.
Figure out the expected vs observed.
In science we like to say around a 95% confidence level.
204
Why are flies (drosophilia) good to study linkage and mapping?
- because it only has 4 chromosomes not 23 like humans, because humans have more, they are harder to study.
205
What is recombination frequency?
- the relative distance between two genes is reflected in the recombination frequency
- farther apart the genes are, they are more likely to crossover and produce recombinants.
206
What is the measurement of Genetic map unit (centimorgan)?
1 genetic map unit = the frequency at which one out of 100 meiosis products is recombinant.
