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«Joshua Frederick Coulcher UCL Submitted for the Degree of Doctor of Philosophy September 2011 Declaration I, Joshua Frederick Coulcher, confirm that ...»

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Shown below are the solutions and media that were used in different experiments described above. Small volumes of solutions were filtered with Millex GP

0.22µm filter attached to a 50ml syringe. Larger volumes were filtered with a Nalgene disposable filter unit 0.2µm (450-0020) attached to a vacuum pump (Gast, DAA-V174ED). pH was measured (where indicated) with a pH meter (Fisherbrand, Hydrus 300).

Bacterial media was autoclaved.

Blocking solution:

Western Blocking Reagent solution (Roche, 11921673001) diluted in PBS-T (1:4)

Carbonate buffer (2x):

120mM Na2CO3, 80mM NaHCO3, pH 10.2

Denaturing RNA gel:

TAE buffer with guanidium thiocyanate

Dinitrophenyl (DNP) RNA labeling mix (10x):

DNP-11-UTP (250nmol, 10mM) (Perkin Elmer #NEL555) Ribonucleoside Triphosphate Set (100mM NTPs) (Roche #1 277 057)

Embryo wash buffer:

0.1M sodium chloride, 0.0004% (v/v) Triton X-100

EGTA:

0.5M pH8.0 Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (Sigma EFast Red:

SIGMA FAST Fast Red TR/Naphthol AS-MX Alkaline Phosphatase Substrate Tablets Set red Staining Buffer (Sigma F-4523)

Fast Red staining buffer:

0.1M Tris (water)

Fixation Buffer (Tribolium):

1.3x PBS (178mM NaCl, 3.5mM KCl, 1.95mM KH2PO4, 8.45mM Na2HPO4, pH7.4) 67mM EGTA, pH8.0

Fixation Buffer with 9.25% formaldehyde (Tribolium):

75% (v/v) Tribolium fixation buffer, 25% (v/v) 37% formaldehyde (Sigma, F8775)

Heparin:

10mg Sigma (H-3393) dissolved in 1ml water.

HMDS:

Hexamethyldisilazane (Sigma, 33011)

Hoyer’s medium:

54g Gum Arabic (Sigma, G9752), 360g Chloral hydrate (Sigma C-8383), 36g Glycerol (28.6ml) 90ml distilled water.

Hybridization Solution:

50% Formamide (v/v), 5X SSC (750mM Sodium Chloride, 75mM Sodium Citrate),

0.1mg/50ml Salmon sperm DNA, 0.05mg/50ml Heparin, 0.02% Tween-20. Made up to 50ml with water and pH adusted to pH6.5 with concentrated Hydrochloric acid.

Inactivation buffer:

Hybridization buffer with 0.3% (w/v) Sodium Dodecyl Sulphate (SDS)

Lithium Chloride:

4M Lithium Chloride (Ambion, AM9480)

NBT/BCIP:

18.75 mg/ml nitro blue tetrazolium chloride and 9.4 mg/ml 5-bromo-4-chloro-3indolyl-phosphate, toluidine-salt in 67% (DMSO) (v/v) (Roche, 1 681 451 001)

NBT/BCIP Staining Buffer:

Tris 100mM pH9.5, NaCl 100mM, 0.02% (v/v) Tween

Phosphate buffered Saline (PBS):

137mM NaCl, 2.7mM KCl, 1.5mM KH2PO4, 6.5mM Na2HPO4, pH7.4

PBS with Tween:

137mM NaCl, 2.7mM KCl, 1.5mM KH2PO4, 6.5mM Na2HPO4, pH7.4, 0.02% (v/v) Tween PEMS (pH6.9)

0.1M Pipes, 2mM MgSO4, 1mM EDTA, pH6.9 Proteinase K 10mg dissolved in 1ml nuclease free water (Sigma, P-2308)

Salmon testes DNA:

salmon sperm DNA (Sigma, D-1626) was fragmented by heating at 95oC for one hour.

Sodium acetate:

3M pH5.2 for molecular biology (Sigma, S7899)

SSC (20x):

3M NaCl, 300mM Sodium citrate.

Stop solution:

0.2M sodium acetate, pH 6.0

TAE buffer (x1):

40mM Tris, 20mM acetic acid, and 1mM EDTA

Torula yeast RNA:

10mg/ml Torula yeast RNA (Sigma, R6625)

–  –  –

Table A.4 Sequence of primers used to amplify the Achaearanea homologue of cnc using RACE.

RACE primer sequence 5' RACE At cnc 1 CCTCGCCGACGAATATCTCTTATCAGAGT 5' RACE At cnc inner 1 TGAGCCTCTGTCAATTCATATTTTGAAAGTC 3' RACE At cnc 1 GACTTTCAAAATATGAATTGACAGAGGCTCA 3' RACE At cnc inner 1 ACTCTGATAAGAGATATTCGTCGGCGA 3’ RACE At cnc 2 GACTTTCAAAATATGAATTGACAGAGGCTCA 3’ RACE At cnc inner2 ACTCTGATAAGAGATATTCGTCGGCGA 5' RACE At dll TGTGCGCGGTTTTCTCATTTTCTTCC 3' RACE At dll ATGTCCATCACCTCCAAGGGACGAT 5' RACE inner At dll ATCGTCCCTTGGAGGTGATGGACA 5' RACE inner At dll GGAAGAAAATGAGAAAACCGCGCACAA 5' RLM-RACE Outer primer GCTGATGGCGATGAATGAACACTG 3' RLM-RACE Outer primer GCGAGCACAGAATTAATACGACT 5' RLM-RACE inner primer CGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG 3' RLM-RACE inner primer CGCGGATCCGAATTAATACGACTCACTATAGG

Appendix 3: Details of Tc cncRNAi experiments

Shown below are the details of Tc cncRNAi experiments performed in chapter four. Table A.5 shows the proportion of Tc cncRNAi knock down phenotypes compared to wild type phenotypes observed during the course of a parental RNAi experiment.

Table A.6 shows the correlation between the concentration of injected Tc cnc dsRNA and mortality of injected Tribolium pupae. Table A.7 shows the numbers of injected Tribolium pupae and the concentration of injected Tc cnc dsRNA used for in situ hybridization experiments.

–  –  –

Tc cnc dsRNA concentration is unknown for this experiment, but is likely to be 1-2µg/µl based upon observed size of RNA pellet during the dsRNA precipitation step. Female pupae injected: 218. Female beetles eclosed: 195. Survival rate from injection to eclosure: 89.4%. Dead beetles by day 20 : 117 (probably female as females were injected with Tc cnc dsRNA which is likely to be responsible for the increased mortality observed). Tc cnc knock down phenotype is characterized by the transformation of the mandibles into maxillae. More extreme phenotypes (failure of germ band formation) were present but not counted.

–  –  –

Mortality assuming that dead adult beetles were injected females as females were injected with Tc cnc dsRNA which is likely to be responsible for the increased mortality observed.

–  –  –

Shown below are the details of Tc DfdRNAi experiments performed in chapter five. Table A.8 shows the numbers of injected Tribolium pupae and the concentration of injected Tc Dfd dsRNA used for in situ hybridization experiments. 3-4 µg/µl dsRNA of Tc Dfd was injected into 80 females to obtain larvae with knock down phenotypes for cuticle preparation.

–  –  –

Shown below are the details of parental RNAi experiments in Tribolium that were performed. Table A.9 shows the numbers of injected Tribolium pupae, the identity of the gene and the concentration of injected dsRNA and observed results of the RNAi experiments.

–  –  –

Shown below are the CNC and bZIP family member genes from different animal species that were aligned with the sequence obtained by degenerate PCR and RACE in the spider Achaearanea, as described in chapter six. Sequences were aligned in order to determine whether the cloned sequence was a true homologue of cnc. Sequences were both manually aligned using Bioedit and aligned with the programs Maclade and ClustalW. cnc homologues are listed in Table A.10. bZIP family members are listed in Table A.11.

–  –  –

Shown below is the cloned sequence of the homologue of cnc in Achaearanea tepidariorum (At cnc), as described in chapter six, that was obtained by degenerate PCR and RACE and then used to make labelled RNA probes to use in in situ hybridization experiments.

At cnc nucleotide sequence obtained from degenerate PCR (72bp):

AGACTTTCAAAATATGAATTGACAGAGGCTCAAATTACTCTGATAAGAGATATTCGTCGGCGAGGAAAAAAT

At cnc nucleotide sequence (1207bp):

CACTCTGTTCCTAAGGCAAATGGATACAACTGTTCAAATTTCTTGCAGCATAATCATACTTACCACATACCCTTCAACAATGATGATCTATGTCACA

AACCAATTACAAGAGACAAGCAAAGATCATATTCTGATGATGATTCTAGCAACAAGGATGAAAAAAGGGCAAAGGAACTCAAACTCCCTATCAG

TATTTCTGATATAATTAATTTAGCCATTGAAGAATACAATGAGAGACTTTCAAAATATGAATTGACAGAGGCTCAAATTACCCTGATAAGAGATAT

TCGTCGGCGAGGAAAAAATAAGGTTGCAGCACAGAACTGTAGAAAACGTAAGATGGATCAAATTAATAACTTGCAACAAGACCTTGATTACCTA

CAAGATGAAAGGATGAAATTGAAATCCAAGCAAAACCATCTGTTGCAGCAAAAGCATGCTATTGAAGAAAAATATGTCCAATTACATGATCTTAT

TTTACAAAATACTAACTGTAGACCACCTAGTGCACCAAGTCTCTCAGCTTTCCATAATTCTCAGAATGCTACATCTGACACTATGTTAGGTAACACT

GACTCCAATGTAAACGGTGGAGCTAAAACAAAAAAGAAATTTAAAAAGTGATAATACATCTATAATTTATTAACCGAATGTGATATACAATTTCTT

ATCGTTAACTACATTTTGATGCCACTCATTTTCATCCATTTATATCATTTGGGAATCATTAAATTTTTTCTTCTTCTAATACATTAATCTGTTAATGCA

TAGATTAAAAAAGTATTTAATGATACTATATCTTTATATTTGTGTTATATTTTATCTTGTACTAAATCACTTTTCTAATAGTACAGTTTTAAGTTGTTA

CTCGCATTTTGTTTTATATTACTTGTATAAATTATGGATCTAAATTGACTTGGCAACAAACAATTTTCATTTAATTTTAATCATTTTACCTCAATCAAT

TTATTTCTTCATAATTATACATAGTTTTTATTTATATTGTTTTCTCTGTTATTGGAATATATTATACAATGGTGTCCTCTGTAATTTTTTTTTCTTTTTA

ATTTTGCATGATTTTTTGATTTGCAAATGATGCCTTATATTTATTTGATTAAACTGTTTTGTTTTCTAATTTGCTATTTTGAAAAGAAAAACATTGTTA

CTTAAACCTTTTTTGATAAAGAAAGAAAATAAAAATATCAGA

At cnc amino acid sequence (207aa):

HSVPKANGYNCSNFLQHNHTYHIPFNNDDLCHKPITRDKQRSYSDDDSSNKDEKRAKELKLPISISDIINLAIEEYNERLSKYELTEAQITLIRDIRRRGKNK

VAAQNCRKRKMDQINNLQQDLDYLQDERMKLKSKQNHLLQQKHAIEEKYVQLHDLILQNTNCRPPSAPSLSAFHNSQNATSDTMLGNTDSNVNGG

AKTKKKFKK



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