Molecular Biology and Development Flashcards
(196 cards)
1
Q
Why is the structure of DNA special?
A
Phosphate molecules are negatively charged
Carbon atoms in deoxyribose are numbered 1’ to 5’
DNA strands are antiparallel
Hydrogen bonds form between base pairs:
A-T 2 H+ bonds
C-G 3 H+ bonds
2
Q
How is the structure of RNA different to the structure of DNA?
A
Ribose is used instead of deoxyribose
uracil is used instead of thymine
3
Q
Which end of the DNA strand is the template strand usually?
A
the 3’ end
4
Q
What are the two ends of polypeptides?
A
Amino/N-Termius or Carboxyl/C-Termius
5
Q
What are the three keys of specificity?
A
Base pairing
Genetic coding - triplet codons
tRNA
6
Q
Describe the role of tRNA
A
tRNA binds to specific amino acids
tRNA recognises corresponding codons in mRNA base pairing
7
Q
Give both the start codon and stop codons
A
AUG
UAG, UGA, UAA
8
Q
What are the modified bases tRNA uses?
A
D, T, Y and Ψ (Psi)
9
Q
What shape is tRNA?
A
“Clover shape”
10
Q
Describe the ‘wobble’ base pairing
A
This is were tRNA can recognise more than one codon at the third position, due to:
G-U forming 2 H+ bond
Adenine forming Inosine (base able to pair with U,C and A)
11
Q
What is aminoacyl-tRNA synthetase?
A
The enzyme used to join the amino acid to the corresponding tRNA using 1 ATP to AMP + 2 Pi
12
Q
What are the two subunits of ribosomes called?
A
Large subunit and small subunit
13
Q
What are the three sites of the ribosome?
A
A, P and E sites
14
Q
Briefly describe the protein synthesis
A
- Messenger RNA bind to the ribosomes
- Aminoacyl-tRNAs recognise and bind to base triplets in mRNA
- Ribosomes transfer amino acids to the growing polypeptide
- The protein is extended from the N-Termius to the C-termius
15
Q
Describe the initiation mechanism
A
- Methionyl-tRNA binds to the P site in the small subunit
- mRNA then binds to the small subunit
- The tRNA anticodon then binds to the start codon on the mRNA
- The large subunit then binds
- Aminoacyl-tRNA for the second amino acid then binds to the A site
16
Q
Describe the elongation mechanism
A
- The next aminoacyl-tRNA recognises and binds to the mRNA triplet at the A site
- The ribosome then transfers the polypeptide from peptidyl-tRNA in the P site
- The large ribosomal subunit moves along relative to the subunit
- The small unit is then moved across, “resetting” the ribosomes. tRNA is also ejected from the E site during this step.
17
Q
What are the roles of elongation factors?
A
EF-Tu - Carries aminoacyl-tRNA to ribosome along with GTP which is hydrolysed to GMP + 2Pi
ED-G - carries GTP to the A site of the large subunit and hydrolyses it to “reset” the ribosome
18
Q
Describe the termination mechanism
A
Translation ends at the first in-frame stop codon
- A release factor protein binds to the A site, instead of tRNA
- The peptyl-tRNA bond is hydrolysed, releasing the protein
- Ribosome dissociates again
19
Q
Describe the “One gene - one enzyme” hypothesis
A
Each gene controls the production, function and specificity of a particular enzyme.
20
Q
Define Genome
A
The whole length of an organism’s DNA - Genes plus non-coding sequences.
21
Q
How many genes do bacteria and animals usually have?
A
Bacteria - ~4,000 genes
Animals - ~20,000 genes
22
Q
Describe a Operon
A
A cluster of genes that are transcribe from one promoter, usually have similar functions.
23
Q
Name a potential problem with translation initiation in operons.
A
-Methionine is found in the middle of proteins, but isn’t the start codon, but translation has to start at more than one place.
24
Q
Sites of translation initiation in eukaryotes
A
First AUG in mRNA.
25
Sites of translation initiation in Bacteria
3' end of rRNA binds to a Shine-dalgarmo sequence in mRNA near the start codon - Sequence similar to AGGAGG.
26
Describe the promoter and it's function
A sequence that provides a binding site for RNA polymerase and specifies the start point and direction of transcription.
27
Describe what a transcript terminator is
Specifies when transcription ends.
28
What does bacterial RNA polymerase consist of?
A core enzyme of five polypeptides - α (2 copies), β, β´, ω.
| But core enzyme cannot recognise specific promoter region so requires sigma factor to form a holoenzyme.
29
How does bacterial translation terminate?
1. mRNA folds into a stem-loop structure followed by a succession of uracils.
2. This structure destabilises the RNA-DNA interactions.
3. RNA polymerase dissociates from the DNA.
30
Give two reasons why genes must be expressed
- When it's product it needed.
| - When a substrate is at the correct level.
31
What is the function of the lac Operon?
To allow E.coli to use lactose as a energy source.
32
How is lactose metabolised in E.coli?
1. Lactose is taken up via the carrier protein lactose permease.
2. β-Galactosidase then hydrolyses lactose to glucose and galactose.
33
Name three genes involved in the lac Operon
lacZ - β-Galactosidase
lacY - Lactose permease
lacA - β-galactoside transacetylase
Arranged like Z,Y,A
34
When is the lac Operon activated?
When glucose is absent but lactose is present.
35
What does a + and - mean over named alleles?
+ - Wild type allele
| - - Mutant allele
36
What does the gene LacI produce?
The repressor regulatory protein for the lac operon.
37
What is a operator? (in terms of the lac operon)
A sequence located just before the lacZ gene where the regulatory protein binds. The operator has a symmetrical sequence. Two Lac repressor polypeptides bind to this and block passage of RNA polymerase.
38
What is allolactose?
Allolactose is a isomer of lactose and is a signal molecule which causes the repressor protein to dissociate from the operator. Formed from the very few molecules of β-galactosidase in the cell. Positive feedback occurs here.
39
Where are the two auxillary operators?
- O2 410bp downstream, in lacZ
| - O3 83bp upstream
40
How does having the auxillary operators affect the rate of transciption?
If O1 is binded with O2 or O3 then transcription is reduced by x1000, as binding is cooperative
41
How does O2 and O3 reduce transcription rate?
- O1 and O2 form a loop in the LacZ gene
| - O1 and O3 form a loop in the promoter region
42
Define "cis"
A cis-acting mutation only affects expression of a gene or operon that is in the same DNA molecule.
43
Define "trans"
A trans-acting mutation can affect expression of a gene or operon in a different DNA molecule. The regulartory sequence does not need to be attached to the target gene or operon.
44
Regulation of the lac operon via glucose
- uses cAMP
- when glucose present cAMP low, when absent cAMP high
- cAMP binds to catabolite activator protein, which is a dimer.
- Protein activates, activating transcription
45
What promoters does the lac operon use?
-35 -10
Standard TTGACA TATAAT
Plac TTTACA TATGTT
PlacUV5 TTTACA TATAAT
46
How does catabolite activator protein increase transcription?
It binds to the DNA next to the -35 promoter region and bends the DNA at a 90 degree angle, RNA polymerase alpha subunit then binds onto CAP and increasing binding of RNA polymerase to the promoter.
47
What are catabolic operons?
- Contain genes for breakdown of metabolites
| - Only activated when metabolite is present and glucose is absent
48
What are biosynthetic operons?
- Contain gene or enzymes of biosynthetic pathways
| - Only activated when end product supply is low
49
What is the trp operon?
- Contains genes for the biosynthesis of the amino acid tryptophan
- Tryptophan repressor protein represses transcription when conc is too high, tryptophan binds with the protein
50
Where is Bioluminescence seen in nature?
Bobtail squid (Eupryma scolopes) contain Vibrio fischeri bacterium, which live in a light organ and in high high concentrations (10^10 cell/ml) light is emitted.
51
How does Quorum Sensing measure cell density?
- LuxI protein catalyses synthesis of an acyl homoserine lactone (AHL), whcih diffuses out of cells.
- Bacteria also take up AHL, when enough bacteria produce AHL, conc outside cell rises inside cells
- Once AHL starts to diffuse bak into the bacterium, it binds with LuxR activating transcription of the lux operon
- AHL binds to the lux box, allowing for positive feedback to occur
52
Where is Quorum Sensing occur?
- Free-living bioluminescent bacteria
- Pathogenic bactieria
- Biofilm formation
53
How is Quorum Sensing used in pathogenic bacteria?
Use Quorum Sensing to only produce degradative enzymes only when density of bacteria is high, activate gene coding for cell wall degrading enzymes, proteases and enzymes for anibiotic synthesis.
54
Define Genomics
The study of genome organisation and the identification of genes and their functions.
55
What was the first eukaryotic genome sequenced?
Yeast (Saccharomyces cereveisiae) - 1996
- 12.1 Mbp in 16 chromosomes
- 5885 likely protein-coding genes
- Approx 140 genes for ribosomal RNA, 275 for tRNA and 40 small nuclear RNAs
56
What was the first plant genome sequenced?
Arabidopsis thaliana - 2000
- 125 Mbp
- 25,498 protein coding genes
57
What was the first two invertebrate sequences?
- Caenorhabditis elegans - 97.1 Mbp - 719,000 genes
| - Drosophila melanogaster - 180 Mbp, 120 Mbp is euchromatin - 13,600 genes
58
What was the first mammal genome sequenced?
Human genome was started in 2000 with a draft and later finished in April of 2003
- 3286 Mbp
- 24,000 genes
59
How is RNA processed in eukaryotes?
- Addition of 7-methyl G cap
- Removal of introns
- 3' end cleavage and addition of poly(A)tail
60
Define Intron
Sequence within the transcribed region of gene that is removed during RNA processing
61
Define Exon
Sequence that is present in mature mRNA
62
Give some characteristics of a Intron region
- Either starts with a GU or a AG
| - Usually has a A next to multiple Y bases in the middle
63
How does RNA splicing occur?
- snRNPs bind to the primary transcript
- Interactions between the snRMPs form spliceosome
- Spliceosome cuts the 5' end of intron and forms a lariat
- Spliceosome cuts the 3' end of intron and joins the exons
64
Define alternative splicing
-60% of human genes coe for more than one protein
-Different exons are used in different kinds of cells
E.g α-tropomyosin: (8 different RNAs)
- helps to regulate muscle contraction
-stablises actin filaments in the cytoskeleton
65
What is repetitive DNA? (Hard one this)
- DNA that occurs in many copies in the DNA
- Amount varies among organisms
- makes up 75% of human genome
66
What types of repetitive DNA are there?
- Simple sequence repeats - ACACACACACACACACAC maybe present in millions of copies, make up ~5% of human genome
- Transposons - 40% of genome, molecular parasites which can duplicate in the DNA.
67
What are the two major classes of Transposons?
- LINES - long interspread elements ~4Kbp (can self replicate as have code for enzymes required)
- SINES - short interspread elements ~100-400bp
68
How do Transposons replicate?
- Uses a RNA intermediate
- RNA polymerase copies DNA to RNA
- RNA is copied to DNA via reverse transcription catalysed via reverse transcription
69
What are Alu elements?
- They are a SINE
- Make up ~10% of DNA
- 71 million Alu elements in human genome
- Non-autonomous
70
Give a example of a compact genome
Yeast - 4% repetitive DNA
- Avg 2Kbp per genome
- ~70% of genome consist of open reading frames
- Little space between genes
- 96% of genes have no introns
71
Give a example of a non-compact genome
Humans - <2% codes for protein
- Avg ~90KB for gene
- Genes commonly have multiple introns, which are longer than the exons
- Avg primary script is ~30Kbp but Avg coding sequence is ~1.4Kbp
72
What types of human globin genes? (finish)
```
: pseudogene
: mainly embryonic
: mainly foetal
(low levels only)
: ‘adult’ (i.e. active after birth)
```
73
What is Syntheny?
The process of which the order of genes in chromosomes is partly conserved through evolution
74
List three essential sequences for chromosomes
- Replication regions, around 30,000 to 250,000 bp
- Ends of chromosomes must be protected
- Specific sequences to allow centromeres to form sister chromatids
75
What is the structure of chromatin?
- Nucleosomes - 8 polypeptides - 2 copies each of histones: H2A, H2B, H3 and H4
- Histones with a positive charge
- DNA forms 2 loops around the histones
76
List all levels of DNA packing
- Double helix
- "Beads-on-a-string" (attaches to H1 proteins)
- Chromatin Fibre
- Loops
- Condensed chromosome section
- Mitotic chromosome
77
Define Euchromatin
- Parts of the chromosome spread out during interphase
78
Define Heterochromatin
- Parts of the chromosome condensed during interphase
79
What is X-Chromosome inactivation?
- When only one X chromosome is active in female mammals, so one is always condensed and inactive
80
Describe both methods of how chromatin is opened
- Histone modification, E.g acetylation by histone acetylases - addition of acetyl groups to lysine residues, removing lysine's positive charge from the histone, reducing electrostatic forces between histones and DNA
- Chromatin remodelling complexes, a active process using ATP by DNA-binding proteins to partly unwrap and move/remove nucleosomes
81
What are the three types of eukaryotic RNA polymerases?
- RNA Polymerase I - transcribes ribosomal RNA genes
- RNA Polymerase II - transcribes genes coding for proteins
- RNA Polymerase III - transcribes genes coding for small RNAs
82
What is the transcription complex?
A purification of eukaryotic RNA polymerases gives core enzymes, which do not initiate transcription specifically, a transcription complex of 50 polypeptides is required for transcription in vivo
83
What does specific initiation require?
- Enzymes to alter chromatin structure
- Specific activator proteins
- Basal/general transcription factors to help RNA polymerase bind to promoter and initiate transcription
- TFIIA, TFIIB for RNA polymerase II
84
What is the key sequence for initiation?
The TATA box is usually the start point of transcription, is 8bp of A or T and is located around -25bp.
85
How does the TATA box initiate transcription?
TATA box binding protein binds to the box and bends the DNA at a ~70 degree angle.
86
How is DNA prepared for transcription?
- Transcription activator proteins binds to specific sequences in the DNA
- Histones are acylated and chromatin is remodelled
- additional activator proteins
87
How is the transcription complex formed?
- TFIID binds to promoter (TBP + TAFs) binding of TFIIA and TFIIB stabilises the complex
- RNA polymerase II and TFIIF bind, forming core pre-initiation complex
- Binding of TFIIE and TFIIH completes the complex
88
How is RNA polymerase released?
- DNA is opened forming a transcription bubble, requires ATP as hydrolysis by TFIIH/ TFIIH also phosphorlytes Pol II
- Transcription starts, but commonly pauses
- Initiation factors dissociate. Elongation factors join with RNA polymerase for elongation
89
List 4 promoters without a TATA box
Element Consensus sequence General TF
- BRE G/CG/CG/ACGCC TFIIB -35
- TATA TATAA/TAA/A TBP -30
- INR C/TC/TANT/AC/TC/T TFIID TSP
- DPE A/GGA/TCGTG TFIID +30
90
What are cis-regulatory elements?
-DNA sequences that form part of a gene and involved in it's regulation, can increase/decrease basal level of expression (some work as more of a on/off switch called response elements)
91
What does the human metallothionein do?
- Binds to metal ions to either protect it form toxins or to store useful ions.
- Transcription induced by metal ions and glucocorticoid hormone.
92
What response elements are found in the metallothionein gene?
- cis-regulatory genes- TATA box
- Sequences increasing expression- GC, BLE, TRE, E-box
- Response elements- MRE and GRE
93
What TFs does metallothionein require?
- AP1 for BLE
- AP2 for TRE
- SP1 for GC box
- USF for E-box
94
Define core promoter, proximal promoter and Enhancers
- The promoter where the RNA polymerase binds, different core promoters contain combinations of defined "elements"
- The region near the core promoter containing cis-regulatory elements
- Cis-regulatory elements far from core promoters
95
Describe how cis-regulatory elements work
1-cis-regulatory elements are recognisable by similarities to a consensus sequence, typically ~10 bp
2-This consensus sequences are recognised by activator/repressor proteins that bind to the specific sequence.
-Either of these steps could alter chromatin structure or interact with transcription complex
96
What are the two classes of hormones?
- Bind to receptors on the outside of the cell and exert their effect indirectly. E.g Peptide hormones - insulin and glucagon (requires intracellular secondary messenger)
- Enter the cell and exert their effects directly E.g small molecules that can easily cross hydrophobic structures
97
List 5 major types of hormones
- Adrenal steroids
- Steroid sex hormones
- Vitamin D
- Thyroid hormones
- Retionic acid
98
What is cortisol used for?
-Released during starvation or intense exercise, binds to glucocorticoid receptor to: Increase blood sugar and has anti-inflammatory and immuno-suppressive action
99
What happens when Glucocorticoid binds to a Glucocorticoid receptor?
- Liver cells- Cortisol increases glucose production form gluconeogenesis form amino acids and other small molecules
- Activates transciption of Tyrosine aminotransferase and PEP carboxykinase
100
What are Ligand responsive transcription factors?
TFs that bind to DNA, they interact with the transcription complex to regulate transcription and are made up of domains with distinct functions
101
What is the domain structure of glucocorticoid receptors?
- ~800 amino acids
| - 1 region transcription activation, 1 DNA binding site and 1 hormone binding site
102
Describe the mechanism of transcription activation by glucocorticoids
1- In absence of hormone, a protein called Hsp90 keeps the glucocorticoid receptor in the cystol, Chaperones the receptor
2- Glucocorticoid hormones enters the cell via diffusion
3- Hormones binds to GR in the cystsol, causing Hsp90 to dissociate
4- GR contains nuclear localisation signal, which is masked by Hsp90, moves into nucleus
5- Receptor binds to glucocorticoid response element in genes, e.g tyrosine aminotransferase and pep carboxykinase, and activates transcription
103
List two functions of Zinc fingers
- Bind of DNA - amino acids in the recognition helix interact with bases in DNA
- Dimer formation
104
What are the response elements for Steroid hormone receptors?
- Similar DNA sequences but GRE is replaced with ERE
| - GGTACA/NNN/TGACCT
105
Describe functions of AP1
- Increases general chromatin accessibility, helps GR bind to GRE
- Insensitive cells don't have hormone receptor
106
Which transcription factors are involved in muscle development?
- MyoD
- Myogenin
- Myf5
- Myf4
107
Define somites
-Segmented block of cells that form along the sides of the notochord in vertebrate embryos. Form vertebrates, ribs, muscles and limbs
108
Describe the formation of muscle
- Gains
- Signals from neighbouring cells induce transcription of MyoD and Myf5 in undifferentiated somite cells, converts somite cells into myoblasts (remodel chromatin)
- Myogenin gene is transcribed in myoblasts, making then fuse and form myotubules
109
How are Induced pluriopotent stem cells formed from Fibroblasts?
-Four TFs - Oct4, Sox2, Klf4 and Myc - can reprogramme fibroblast into IPS cells
110
How are Islet B-cells produced?
-Three TFs - Pdx1, Ngn3, MafA
111
Regulation of translation and mRNA stability
Iron regulates:
- translation of mRNA for iron storage protein ferritin, when iron concentration is high ferritin mRNA is translated, it's cis-regulatory element
- Stability of mRNA for iron uptake protein transferrin receptor, when iron concentration is low, mRNA is more stable
112
What were the three original theories of evolution?
- Preformation theory - Everything in the embryo is performed and gets bigger as it develops. "Homunculus" - Little human embryo
- Epigenesis - New structures arise by progressing through different stages
113
Describe Cell Theory
(Mattias Schledien and Theodor Schwann 1838)
- Organisms are composed of cells, the basic unit of life
- Multicellular organisms are made from single cells, fertilised eggs
114
What is The germ line concept?
August Weismann (1834-1914)
- Only the germ cells pass on characteristics to the offspring
- Somatic cells are not directly involved in passing on traits to the next generation and characteristics acquired during a animal's life aren't
- Body is germ cells
- Zygotes contains the physical basis of heredity
115
Define the terms "Descriptive" and "generative"
- Descriptive - Blueprint/Plan, contains detailed description of a object
- Generative - Describes how to make an object
116
List the five processes in development
- Cell Division - Cleavage
- Pattern formation - Spatial organisation of cells
- Morphogenesis - Changes in form
- Differentiation - Cell specialisation
- Growth
117
List the five key cell activities
- Cell-Cell communication
- Cell shape changes
- Cell movement
- Cell proliferation
- Cell death (apoptosis)
118
Define all positional key terms e.g Cranial
```
Cranial- cephalic (head)
Rostral- nasal Region
Caudal- tail
Medial- near the midline
Lateral- to the side
Apical- at the apex
Basal- at the base
```
119
What do the three germ layers differentiate into?
- Endoderm - Gut, Liver and Lungs
- Mesoderm - Skeleton, Muscles, Kidneys, Heart and Blood
- Ectoderm - Epidermis of skin, Nervous system
120
What are the two types of cell fates? (Hard)
- Mosaic development depends on specific 'determinants' in the zygote that are distributed unequally to the daughter cell via cleavage. The fate of each cell is predetermined in the egg by the factors it receives during cleavage. The cell's environment cannot influence it.
- Regulative development depends on interactions between 'parts' of the developing embryo by cell to cell communication. The cell's environment can influence it's development fate. The embryo is able to develop normally even when parts of it are removed or rearranged.
121
Evidence for Mosaic development
```
Wilhelm Roux (1888)
-Destroyed one cell in a two cell embryo, he concluded that frog development is moasic, so taht the cells have their character and fate determined by each cleavage as only half the embryo developement.
```
122
Evidence for Regulative development
```
Hans Driesch (1891)
-Sea urchins embryos could be seperated when it is a two-cell embryo and still grow into a slightly smaller but normal larva.
```
123
Define Cleavage
A series of rapid cell divisions after fertilisation without growth that divides the embryo up into a number of smaller cells.
124
List the egg axes
- Non-yolky - the animal pole
| - Yolky -the vegetal pole
125
Explian what occurs during the reorganisation of the egg cytoplasm
- Egg loses it's radial symmetry.
- Cytoplasm streams towards vegetal pole.
- Bilateral symmetry establishes at the left and right side of embryo.
- Grey crescent is formed in amphibian eggs.
- Yellow cytoplasm of tunicate eggs.
126
What was found in Conklin's study (1902)?
-Cells which contain yellow cytoplasm after cleavage form muscle cells, supporting the germ line concept.
127
List the three patterns of cleavage
- Radial cleavage - Deuterostomes
- Spiral cleavage - Protostomes
- Superficial cleavage - insects
128
Define 'Holoblastic cleavage" and "Meroblastic cleavage"
Holoblastic cleavage - complete cleavage, all cells in embryo become separate.
Meroblastic cleavage - Incomplete cleavage, only see cells in some parts of the embryo.
129
Why is the Nematode a good model organism for development?
- 1mm long
- Easy to see cells with nomarski microscopy
- 15h embryogenesis
- 959 somatic cells
- 131 cells die during development
130
Which cell in two-cell embryo is the largest?
-AB cell, ABa and ABp change fate according to their position.
131
What other use does cleavage patterns have?
-They are able to help form a fate map of all of the cells in the body.
132
What are P granules in cytoplasm?
-A mixture of RNA and protein, which after fertilisation become concentrated at the posterior.
133
Define Gastrulation
-Formation of gut and main body plan.
134
Describe Enchinoderm Gastrulation
- Vegetal pole of the bastula flattens
- Some cells change shape and move inward to form a archenteron
- Other cells break free, becoming primary mesenchyme.
- More cells break free forming the secondary mesenchyme, thin extensions of these cells (filopodia) attach to the overlying ectoderm.
- The archenteron is elongated by contraction of mesenchyme filopodia and cell rearrangement
- The mouth will form where the archenteron meets the ectoderm.
- The blastopore will form the anus of the mature animal
- Primary mesenchyme will form spicules, early form of skeleton.
135
What is the pattern of radial cleavage?
-1-cell to 4-cell to 8-cell to (unequal 4th cleavage) 16-cell to 32-cell to 64-cell (bastula)
136
What happens when you split a embryo at the 4-cell stage?
- Animal half of the embryos give rise to 'animalised' embryos, which contain structures related the ectoderm.
- Vegetal half embryos give rise to 'vegetalised' embryos, which contain structures skeletal structures and gut strcutures.
137
Why are micromeres considered special?
-Because they can influence cells around them as when 4 are added to a Animal half embryo, a gut is formed and a normal larva (pluteus) is formed.
138
How many micromeres are needed?
- Animal 1 layer + 4 micromeres = normal pluteus
- Animal 2 layer + 4 micromeres = vegetal larva, archenteron is over-expressed
- Animal 2 layer + 2 micromeres = normal pluteus
- Animal 2 layer + 1 micromere = Animalised larva, permanent blastula
139
What other factors can effect larvae?
- Li+ ions - vegetalised larvae
| - Many dyes/Zn2+ - animalised larvae
140
What is the gradient theory?
- Positional status of a cell may be defined in relation to 2 gradients of influence - animalising gradient and vegetalising gradient.
- Gradient is usually a morphogen.
141
Describe Lewis Wolpert's french flag model (1960s)
- Each cell has a potential to develop as 'blue', 'white' or 'red'.
- It depends upon the position of the cell in the concentration gradient.
142
Define a Insulator
-A control element that blocks enhancer action, weakening/turning off transcription.
143
What is beta catenin?
- Beta catenin is a TF (for the -pmar1 gene) that is derived from maternal RNA, which is deposited into the egg.
- Beta catenin accumulates in the micromere and veg. cell nuclei.
144
What happens to beta catenin when embryos are treated with lithium chloride?
-Beta catenin accumulates in cell nuclei all over the blastula, causing a vegetalised larvae to be formed.
145
What happens to when beta catenin is prevented from entering the nuclei?
-Beta catenin remains in the cytoplasm and causes the cell to become animalised.
146
What is a GRN?
-A Gene Regulatory Network and Beta catenin works as a TF to deactivate a repressor gene for gastrulation.
147
What is the Endo-16 gene?
-A gene which has 13 TFs that bind to 50 binding sites
148
What is a kernal?
- GRNs that aren't used anywhere else but in development
- Same kernals for endoderm/mesoderm for most organsims.
- Kernals are still present in species today, as species still look the same as they did 500mya.
149
Define 'Oogenesis' and 'Spermatogenesis'
- Oogenesis is the formation of eggs.
- Spermatogenesis is the formation of sperm.
- Both gametes are formed by meiosis.
150
Describe Oogenesis and it's features (Hard)
-The primary oocyte is diploid, but undergoes meiosis to form haploid cells - the mature ovum.
Features:
-Storage molecules 'yolk' containing lipoprotein 'vitellin'
-Membranes and associated structures involved in the fertilisation.
-Mitochondria, ribosomes and stored mRNA.
-Vitellogenesis may occur through a lengthened prophase of meiosis (may be months-years).
-The oocyte increases enormously in size and the reduction division may be delayed until after this growth phase.
151
Describe how oogenesis can be classified using relationships of the oocyte to 'other cells'
- Solitary - no follicle cells or nurse cells, found in many polychaeta
- Follicular - Associated with a covering of somatic cells, found in tunicate Ascidella aspersa and locusts
- Nutrimentary - associated with sister cells (nurse cells), found in Drosophila
152
What type of ovaries do locusts have?
-Panoistic ovaries
153
What do Drosophilia ovaries show?
- Polytrophic meroistic development, which allows the oogonia to divide into both oocytes and nurse cells.
- Incomplete cell divisions occur to leave cytoplasmic bridges between nurse cells and the oocyte.
154
List the ways oogenesis can be classed
- Relationship of oocyte to 'other cells'
| - The synthetic pathway for the stored yolk in the cytoplasm.
155
Describe Autosynthesis
- The synthesis of yolk and other stored materials by the oocyte itself.
- Uptake of low molecular weight precursors of yolk – amino acids and carbohydrates.
- Autosynthesis is sometimes observed in solitary oogenesis in polychaetes.
156
Describe Heterosynthesis
- Synthesis of yolk proteins is done by other, non-germ cells.
- Characterised by uptake of vitellogenin, later stored as vitellin.
- Vesticles seen forming on cell surface and electrophoresis shows proteins identical to yolk protein.
157
What is the 'Masked mRNA' hypothesis?
- During oogenesis, the oocyte accumulates stored RNA, some of which is ribosomal.
- Much of the RNA is mRNA, which 'stores developmental information'.
- mRNA only made available after fertilisation.
158
What did Nusslein-volhard and Wieschaus study?
- Treated flies with chemical mutagen, and found that mutations caused larval segments to be missing.
- Show mutations dependent on genotype of the mother (Maternal genes).
159
List the five main segments of the Drosophila Larva, Anterior to Posterior
- Acron
- Head
- Thorax
- Abdomen
- Telson
160
Which segments do bicoid mutants lack?
- Anterior (Acron, Head and Thorax)
| - Injection of wild-type egg cytoplasm into mutant egg generates head segments, suggests mRNA is translated after birth.
161
Which segments do nanos mutants lack?
-Posterior (Abdomen)
162
Which segments do torseo mutants lack?
-Terminal (Acron and Telson)
163
Describe maternal gene gradients
- Nanos and Bicoid RNA are polar opposites with nanos at the posterior and bicoid at the anterior.
- Hunchback and Caudal RNA is constant across the oocyte.
- Bicoid suppresses translation of Caudal RNA and Nanos inhibits translation of Hunchback.
- Therefore two gradients are formed on the A-P Axis.
164
Explain how maternal mRNAs become distributed in the oocyte
- gurken mRNA is translated in the posterior and protein binds to the torpedo receptor in the posterior follicle cells.
- Causes posterior follicle cells to produce a signal that rearranges microtubules in the egg cytoskeleton.
- Maternal mRNAs from nurse cells are transported along the microtubules.
- Movement of gurken mRNA associated with movement of nucleus to establish dorsal axis.
165
List the 6 gene expression cascade
- Maternal genes
- Gap genes
- Pair-rule genes
- Segmentation genes
- Selector genes
166
Describe Gap genes and give three examples
- Mutations usually cause segmentation deletions.
- These are the first zygotic genes to be shown across a AP axis, code for TFs.
- hunchback is a gap gene, which is activated by bicoid protein.
- Hunchback protein activates/represses other gap genes e.g knirps, kruppel and giant.
167
Describe Pair-rule genes and give two examples
- Earliest segments are 14 parasegments
- E.g Even-skipped and fushi taraza
- Each parasegment is split into two genes equally up the embyro in a alternating pattern.
- Even-skipped stripe 2 is formed when: 1. bicoid and hunchback activate the eve gene. 2. giant and kruppel repress the eve gene at a certain threshold even if bicoid and hunchback are present. 3. Anterior boarder is set by the giant protein and posterior by kruppel.
- ~500bp regulatory region
168
Describe segmented polarity genes and give a example
- Engrailed is a segment polarity gene that is exposed in the anterior region of each parasegment.
- Towards the end of maturation, a frame shift occurs and the mature segments are made up the posterior of the parasegment in front and the anterior of the original parasegment.
- If fushi tarazi gene not expressed then a mutant is formed with too few segments.
- 'denticles' are formed on the ventral surface, dentricles are only produced in the anterior region.
- Engrailed's protein turns on the hedgehog gene.
169
List the segments of a Drosophila
- Head: Mandibular, Labial, Maxillary
- Thorax: T1 (Leg 1, no wing), T2 (Leg 2, wing) and T3 (Leg 3, haltare)
- Abdomen: A1-A8
170
Describe the imaginal disc system
- Adult ectodermal structures develop from imaginal discs.
- Pad of undifferentiated cells in the larva.
- Each disc has it's own fate and fate map.
- Discs can undergo transdetermination, implanting discs into other areas of the embryo will produce mutant flies with 'inappropriate' appendages. Mutants called homeotic mutants.
171
Give three homeotic mutant examples
'Antennapedia' - antennae converted into legs
'Bithorax' - anterior part of haltere converted into a anterior wing
'Ultrabithorax' - both parts of the haltere are enlarged
-Mapped by Ed Lewis
172
What are the two main clusters of hox genes in Drosophila?
-Antennapedia and Bithorax
173
Define a Homeotic gene
-A gene which defines a region or position in the embryo. Mutations in homeotic genes lead to transformations of one structure into another.
174
Define a Homeobox
-A region of DNA in homeotic genes that encodes a DNA-binding called the homeodomain.
175
Define Hox genes
-A family of homeobox-containing genes that are also part of a homeotic gene complex.
176
What is the HOM-C?
-The antennapedia and bithorax gene clusters.
177
Explain the the Bithorax complex and segmentation
- When all hox genes from biothorax complex are deleted parasegments 5-13 turn into segments.
- Ultrabithorax specifies parasegments 5 and 6.
- Abdominal-A specifies parasegments 7,8 and 9.
- Abdominal-B specifies parasegments 10,11,12 and 13.
178
List the 5 target genes for hox proteins, their functions and what they're regulated by
- distal-less - Activates gene pathway for limb formation - ULTRABITHORAX
- distal-less - Activates gene pathway for limb formation -- ABDOMINAL-A
- decapentaplegic - Triggers cell shape change in the gut that are required for normal visceral morphology - ULTRABITHORAX
- reaper - Apoptosis of boundary between the maxilla and mandible of the head - DEFORMED
- dapentaplegic - Prevents the above cell changes in more posterior - ABDOMINAL-B
179
List 4 ways in which hox genes could be linked to body plan
- Changes in gene number.
- Broad changes in hox expression.
- Subtle changes within hox domains.
- Changes in regulation or function of downstream genes.
180
What causes shifting zones of hox genes expression in crustaceans?
-If no Ubx and Abd-A expression, anterior limb/locomotive on thoracic segment is converted into a maxilliped. E.g Lobster on T1 and T2, Brine shrimp expression is constant.
181
Why do butterfly larvae have prolegs on their abdomen?
- This is due to the distal-less gene being expressed in patches of cells in the abdomen.
- Distal-less is usually inhibited by Abd-A and Ubx but they aren't present in these patches.
182
Describe Amphibian gastrulation
- Begins with involution of the mesoderm and endoderm at the dorsal lip and under the ectoderm.
- The endoderm then forms a roof over the archenteron, forming the gut.
- Ectoderm cells spread out from the animal pole, this is called epiboly.
- Mesoderm cells move under the ectoderm, convergent extension.
183
What is Neurulation?
- The inwardly migrating cells form the roof of the archenteron.
- These cells will form the dorsal mesoderm: in the midline notochord (form vertebral column), dorso-lateral the somites (form vertebrae, ribs, muscle and skin)
- Cells above the mesoderm are referred to as the neurectoderm and usually form the brain/spinal cord.
- Neurectoderm first form a neural plate, then a neural groove, followed by a neural tube. This process is called primary embryonic induction.
184
Define 'Determination'
-A stable change in the internal state of a cell such that its fate is now fixed, or determined.
185
Define 'Induction'
-The process whereby one group of cells signals to another group of cells in the embryo and so influences how they will develop.
186
Define 'Primary embryonic induction'
-The induction of the whole body axis.
187
Define 'Organiser'
-A signalling centre that directs the development of the whole embryo or part of the embryo, such as a limb
188
Why is the grey crescent region so important in Amphibian development?
-Because it gives rise to dorsal structures such as a nervous system, notochord and somites.
189
What happened in Spemann's study? (1918)
- Transplanted tissues between newt embryos of different species with differentiation.
- Result depended on age of embryo.
- Fate of transplanted tissue determined in later stages.
190
What happened in Spemann and Mangold's study? (1924)
- Took cells from the dorsal lip and placed them on the ventral side of the embryo.
- Found that a secondary invagination occurred.
- Dorsal lip cells deemed as organisers and cause primary embryonic induction.
191
What is the primary inducer for the formation of neurectoderm? How?
-Beta catenin
1- B-catenin gradient, synthesised via maternal mRNA.
2- Dishevelled protein in vegetal pole is transported by microtubules along cortical cytoplasm to future dorsal region (DSH.
3- Glucose Synthase kinase 3 (GSK-3) normally degrades B-catenin but is blocked by DSH.
4- Therefore ventral side of embryo receives B-catenin while dorsal doesn't.
-B-catenin and TGF-B proteins (from ventral side) overlap in the 'Nieuwkoop centre'.
192
Describe the Neurectoderm GRN
- Siamois gene normally repessed by Tcf-3 TF in ventral side.
- B-catenin activates expression of siamois gene in dorsal side.
- Siamois protein interacts with transforming growth factor-B proteins to control goosecoid transcription.
- Goosecoid protein activates genes in the organsier.
193
Examples of organiser-specific proteins
- Nuclear proteins: Goosecoid, Xnot and XFD1
| - Secreted proteins: Chordin, Dickkopf, Noggin and Sonic hedgehog
194
What is BMP4 and what does it do?
- Bone morphogenetic protein 4 inhibits cells from neural tissues.
- Expressed ventrally during gastrulation.
- Several genes expressed by the organiser suppress BMP4 signalling in dorsal region.
195
What are the three main limb regions?
- Stylopod- Humerus and Femur
- Zeugopod- Radius and ulna, Tibia and fibula
- Autopod- Carpals, Metacarpals, Tarsals, metatarsals and digits
196
Describe limb buds
- Formed from mesodermal cells that form under the ectoderm.
- Half a limb bud can form new limbs.
- In land vertebrates, there are always 4 limb buds per embryo, always opposite.
- Have a Apical ectodermal ridge with a mass of inner mesodermal cells.
