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2

Lecture 1

Genetics and Development.

3

Give 6 examples of model organisms.

S. cerevisiae. Arabidopsis thaliana. C. elegans. Drosophila melanogaster. M. musculus. D. rerio.

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What is C. cerevisiae used for?

Homologous recombination.

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Why is A. thaliana used?

Small genome. Many seeds.

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Why is C. elegans used?

Transparent. Life history of every cell observed by laser ablation.

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What is D. melanogaster used for?

Balancer stocks. Transgenesis. Mosaicism.

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What is M musculus used for?

Transgenesis. Gene targeting

9

What is D. rerio?

Zebrafish.

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What is D. rerio used for?

Embryonic development studies.

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Why is D. rerio used?

Transparent embryo

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Why are model organisms used?

Easier to manipulate than target organism. Information can be extrapolated.

13

Give an example of a gene which is reused in the organism for a different purpose.

Drosophila Notch gene: Neural precursor selection. Muscle precursor selection. Malphigial distal cell selection.

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Give an example of a gene which is used in other organisms for the same purpose.

Pax6 gene: Eye development in humans and Drosophila.

15

Give 2 examples of genes which are used for different purposes in different organisms.

Pax6 used for muscle development in mice but eye development in humans; Toll receptor pathway.

16

How are hox genes studied in development? Give an example.

Studied in drosophila. Homologues cloned and studied in vertebrates. Hox-9/-11/-13 in chick limb bud - Expression regulates additional digit formation.

17

What are the 3 types of mutagenic agents?

Biological. Chemical. Radioactive.

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Give an example of a biological mutagen.

tDNA insertions from agrobacterium.

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Give an example of a chemical mutagen.

Alkylating agents.

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Give an example of a radioactive mutagen.

UV (causes thymine dimers).

21

What are the 6 types of mutations?

Deletions; Additions; Inversions; Point changes; Duplications; Translocations

22

What are the 4 possible effects of mutations? Give examples.

Loss of function - Achondroplasia (Bone proliferation); Conditional mutation - Protein function reduced under certain conditions (Temperature); Gain of function; Expression mutants - Expressed at wrong time/place (Antennapedia).

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What is saturation mutagenesis?

Isolation and epistatic analysis of different mutants to determine the components of the pathway.

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Why may some genes be omitted when performing saturation mutagenesis?

Mutant lethality.

25

What is transgenics?

Introduction of modified gene back into the organism via vector.

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What are the aims of evodevo?

Identification of genes. Understanding of their effect. Determining coordination of gene activity.

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Lecture 2

Determining Genotype from Phenotype.

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Outline the stages of determining genotype from phenotype.

Map mutant gene to a chromosome by linkage mapping with fluorescent markers. Map with deletions/breakpoints within the locus to determine precise gene location. Examine sequence and determine coding sequence. Consider candidate genes based on protein properties and expression patterns.

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How is the physical extent of deletions determined?

Creating a heterozygote and observing the deletion loop size.

30

What are Drosophila polytene chromosomes?

Up to 1024 chromatids joined. No mitosis but replication still occurs.

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What is pseudo-dominance?

Dominance of the recessive allele in absence of the dominant allele.

32

How can a DNA sequence be examined?

By comparing PCR result to human genome project log.

33

How can canditdate gene expression be observed?

In situ hybridiasation; Northern blotting.

34

How can location of an encoded protein be detected directly?

Immunohistochemistry.

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What exactly can be determined from ISH?

Cells expressing protein. Time of expression.

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Give an examplea of label used in ISH.

Digoxigenin on uridines.

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How is digoxigenin marker detected?

By binding antibodies labeled with alkaline phosphate. Add alkaline phosphatase to convert alkaline phosphate to a coloured dye.

38

What is in vitro mRNA synthesis?

Synthesis of mRNA outside of cell. Using phages (T7/SP6) for transcription of mRNA from a template complimentary DNA (cDNA) polylinker.

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What is a polylinker?

A sequence of complimentary DNA with up to 20 restriction sites.

40

What is immunihistochemistry?

Use of antibodies to identify cells which express the target protein and determining the location of protein within the cell.

41

Outline how monoclonal antibodies are synthesised.

Inject purified target protein into mouse. Fuse spleen and myeloma cells to create hybridomas. Culture in hypoxanthine-aminopterine-thymidine (HAT) medium (blocks DNA synthesis to promote cell division). Select for positive cells and harvest monoclonal antibodies from those.

42

Outline how immunohistochemistry is carried out.

Inject antibodies into cell synthesising the protein. Fix cellular contents of tissue sample.Use 2nd, labeled antibody to detect the 1st (Goat ab against mouse Igg). 2nd ab labeled with a fluorochrome or enzyme (HRP).

43

How would you test that the chosen candidate gene is indeed responsible for the mutant phenotype?

Introduced cloned DNA via P element transgenesis (for drosophila), into a mutant organism to check for recovery of phenotype.

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What is a P element?

Class of transposable element. Inserts itself into drosophila genome.

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What is the function of P elements?

Source of mutation/ chromosomal rearrangement.

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What are the properties of P elements?

2.9Kb. Inverted 31bp repeats at either end. 4 exons (code 2 proteins including transposase).

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What is the function of transposase?

Identifies inverted repeats and catalyses recombination with host DNA. Excises P elements to recombine elsewhere.

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How can P elements be used as a trojan horse?

Replace the P element of E.coli (coding sequence only) with BacOR. Add a marker gene (eye colour). Introduce the new "P element" into mutant flies for white gene. Wild type copy of white gene will be linked to the eye pigment of P element (?).

49

Outline how transgenesis experiments are carried out in drosophila.

Modofied P element with associated marker introduced into flies together with a helper plasmid (carrying transposase), before cell walls form. Helper plasmid cant insert into genome - lacks inverted repeats - produces transposase instead. Transposase inserts experimental P element into genome. Plasmids degrade. If the P element inserts into germ cells, next generation expresses the P element's phenotype. Expression tests (immunohistochemistry) carried out on transgenic next generation flies.

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Lecture 3

Drosophila Life Cycle.

51

Outline the stages of Drosophila life cycle and their duration.

Embryo - 1 day; 3 Larval stages - 4 days; Pupal stage - 5 days; Adult fly.

52

Outline the body plan of the drosophila embryo.

A-P segmentation: 3 head; 3 thoracic; 8 abdominal.

53

What defines the polarity of a drosophila embryo?

Protein gradients set up by maternal DNA products (as well as by mRNAs). Translational regulation of zygote's genes leads to repeated pattern of segments.

54

What is the evidence supporting the role of maternal mRNAs in polarity establishment?

No gene expression from zygote's nucleus for first few hours. Maternal mRNAs present at opposite poles. Gradients establish before gene expression from zygote nucleus.

55

What are nurse cells?

Polytenic germline cells of the follicle.

56

What is the function of nurse cells?

300-fold transcription rate of normal diploid cells used for formation of oocyte cytoplasm via ring canals.

57

What is a syncytium?

Multinucleate cell caused by rapid nuclear division with no membrane formation.

58

Describe the nuclear divisions leading to the syncytic drosophila embryo.

9 nuclear divisions. 15 of resulting nuclei form germ cells at posterior. 4 further divisions of the rest. Distribute arounf the edges. Membranes form ~6000 cells.

59

What is gastrulation?

Formation of the 3 germ layers.

60

Describe Klaus Sander's experiments on insect embryos.

Set up experiments by tying down embryos to arrest cytoplasm movement. Injected cytoplasm from A to P to observe change in gradient.

61

What are the effects of exposure of the anterior to UV?

Embryo forms 2 posterior patterns - mirror image.

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How has the presence of RNAs at the anterior of embryo been proven?

Injection of RNAse into anterior causes 2 posterior patterns.

63

What is the function of the bicoid gene?

Transcriptional regulation of anterior development.

64

Describe the phenotypes of various bicoid genotypes.

Homozygote wt and mt both express wt phenotype. Heterozygotes may express mutant phenotype.

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Explain the unusual phenotype expression of bicoid.

Neither wild type nor mutant alleles of bicoid are haplosufficient.

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What are the main segmental regions of drosophila embryo?

Acron (terminus); Head (anterior); Thorax (anterior); Abdomen (posterior); Telson (terminus).

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Describe the phenotype of mutants of the 3 A-P determining pathways.

Anterior - no head/thorax. Double abdomen. Telson instead of acron; Posterior - No abdomen. Expanded head/thorax; Terminal - No acron/telson.

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Describe the patterns of mRNA established in early embryo.

Bicoid mRNA at anterior - sharp gradient; Nanos mRNA travels to posterior to set up its gradient.

69

Sequence-wise, what is required of mRNAs to establish axis? Support.

Specific sequences in 3'UTR regions. Swapping of UTRs caused wrong development.

70

How and why is the BCD protein gradient different from the bcd mRNA gradient?

Anterior to posterior but more diffuse. Set up by variant polyA tail length.

71

What influences the BCD protein gradient?

Rate of diffusion. Uptake into nuclei. Protein half-life.

72

Describe the translational control gradients of the embryo.

BCD represses translation of "caudal" mRNAs causing the caudal gradient to be P-A. NOS represses translation of hunchback mRNAs causing the hb gradient to be A-P.

73

Describe the mRNA gradients of caudal and hb.

Both evenly spread throughout the embryo.

74

How has the nanos binding region of hb been determined?

Hybridisation of hb sequences to lacZ coding region. Hybrid not expressing lacZ must contain the specific UTR since nanos has repressed expression.

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What are the specific nanos-binding UTRs of hb?

GUUGU; AUUGUA.

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nanos-binding UTRs of hb are called NREs. Why?

Nanos Response Proteins.

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How can the repression of nanos at those UTRs be tested?

Splicing of the UTRs into a reporter gene. Injection of gene into embryo. If translation repressed at posterior (nanos location) - UTRs bind nanos.

78

Describe the interactions of hb and bcd.

Bicoid activates zygotic hb. Increasing bcd dosage leads to a steeper hb gradient. Hb requires a threshold concentration of bcd to be activated. Increasing bcd concentration moves the start of gradient towards the posterior.

79

What is the evidence to support involvement of bcd in anterior development?

Mutants show no anterior structures. Expanded posterior structures instead. Injection of bcd into anterior of bcd mutant restores phenotype. Injection of bcd into posterior of wt embryos causes formation of 2 heads in larvae.

80

bcd/hb/nos/caudal are described as morphogens. What are morphogens?

Substances which determine the pattern of morphogenesis. Diffuse from a source. Action is concentration dependant.

81

Give an example of how bcd function is concentration dependant.

Increased bcd in mother causes increased bcd in embryo. Cephalic furrow occurs more posteriorly due to a larger gradient of bcd hence increased development of anterior structures

82

Outline the translational regulation of morphogens by bcd.

Activates translation of zygotic hb. Represses translation of caudal mRNAs.

83

Describe how bcd regulates transcription of hb.

Binds to 5 sites on hb promoter. 9bp each. Some sites have a higher affinity than others.

84

How have the bcd binding sites on hb been identified?

DNA footprinting: hb promoter cloned. One end of DNA labeled with 32P isotope. DNA digested with DNAseI to obtain a ladder of fragments. Fragments form bands on agarose gel (continuous column). Express bcd in bacteria. Bind bcd to 32P labeled hb promoter strands in vitro. Digest sample with DNAseI. Bound fragments protected from DNAseI. Electrophoresis of digested fragments shows gaps in the column. Gaps correspond to the binding sited of bcd.

85

What is the evidence for bcd binding sites to be responsible for regulation?

Fusion constructs: hb promoter spliced into a reporter gene. Introduction of bcd into transgenic embryos containing promoter+reporter. Expression of reporter protein proves that promoter sites are responsible for regulation of hb by bcd.

86

How can high and low affinity bcd binding sites be compared?

Testing of synthetic 9bp binding sited in bcd mutant embryos.

87

Describe the synergistic action of hb and bcd on anterior development.

High concentration of bcd and/or the presence of hb causes expression of anterior development genes (otd/ems/btd). Different target genes respond to different concentrations of bcd.