MATERIAL AND METHODS
2.8 kb cocu enhancer in pattB
The 2.8kb enhancer was amplified using the primer pair D5 F & D5 R, which contain BamHI sites (see Table 2 for primer list). After restriction digest the PCR product was cloned in the BamHI site of the pattB vector. Both orientation of the product were isolated and sent for injection at Genetic Services Inc (Cambridge, Mass) into the VK00001 (59D3) platform (Venken, He et al. 2006).
RNAi experiment.
Fecundity.
We used 3-5 day old virgin females from the w; DI-Gal4 as a specific secondary cell driver line and crossed them to males carrying different shRNA constructs for our candidate
99 genes (see table 6 for the list of genes and fly line ID from the VDRC). From theses crosses, male virgin progeny were collected and aged 3-6 days. Ten single crosses were established for each RNAi line with a single CS female in small vials with food. iab-6cocu males served as positive controls for the cocu phenotype, while w; DI-Gal4 /+ was used as negative control, females mated to these males show normal egg-laying phenotype. The male was removed after approximately 24h and the female was transferred to a new vial. The eggs laid in the previous vial were counted. For a period of five days, the process of female transferring and egg counting was repeated. Average number of eggs per day were analyzed in order to determine if a cocu phenotype was present.
Secondary cell morphological phenotype.
Male progeny from the (♀w; DI-Gal4,UAS-GFP X ♂RNAi and ♀w; DI-Gal4 X ♂RNAi) cross were dissected, mounted in PBS and observed under a fluorescence or light microscope for disturbance in the morphology of the secondary cells. Pictures were taken for every gene knockdown and compared to the cocu secondary cell phenotype.
In situ hybridization of accessory glands.
Probe synthesis.
We amplified the probe sequences for each candidate gene from previously isolated RNA from accessory glands using the “OneStep RT-PCR Kit” (Cat.no. 210210 - QIAGEN), (see table 7 for primer information). The PCR products were gel purified and their concentration determined by nanodrop. Using the “PCR DIG Probe Synthesis Kit” (Roche,
100 Cat.no.11636090910) with only the antisense primer and 100-200ng of gel purified PCR product, DNA probes were synthesized. Unincorporated nucleotides were removed from probe solutions by standard ammonium acetate/ethanol precipitation and probes were rehydrated in water.
Tissue preparation.
Accessory glands were dissected and fixed for 10 minutes using 4% paraformaldehyde in PBT (Tween 20 – 0.01%) followed by 5 minute washes with PBT. Next, they were transferred to 100% ethanol with two intermediary wash steps. First, they were placed in a solution of PBT:
ethanol, 1:1. After 5 minutes, half of the volume was removed and ethanol added in the amount of the removed volume. 5 minutes after that, the incubation solution was removed and the glands were washed 3 X 5 minutes with ethanol. At this step, the tissue was stored at -20°C until needed or at least for an hour before the next step. Rehydration of the tissue was performed by going through a 5 minute wash of 1:1 ethanol:PBT, a second 5 minute wash where you take away half the volume and replace it with PBT, followed by 3 X 5 minute washes of PBT. 2 X 5 minutes rinses were performed with 0.1M Triethanolamine, followed by 2 X 5 minutes washes with 0.1M Triethanolamine supplemented with acetic anhydride (2.5uL acetic anhydride in 1mL 0.1M Triethanolamine – made fresh). Afterwards, the glands were washed 3 X 5 minutes with PBT.
101 Prehybridization.
Prehybridization (50% Formamide, 5X SSC, 0.01% Tween 20), hybridization (50%
Formamide, 5X SSC, 100ng/ul salmon sperm DNA, 50ng/ul Heparin, 0.01% Tween 20), and probe hybridization (50% Deionized formamide, 5X SSC, 100ng/ul salmon sperm DNA, 50ng/ul Heparin, 0.01% Tween 20) solutions were made. Tissues were incubated in a 1:1 mix of prehybridization solution with PBT for 5 minutes. Then incubated for 5 minutes in prehybridization solution. The hybridization solution was boiled for 5 minutes, then cooled on ice for 5 minutes. The hybridization solution was added to the tissue and incubated for at least an hour at 45°C.
Hybridization.
Approximately 1uL of probe solution was added to 100uL of probe in hybridization solution to make the working probe hybridization solution. The working probe hybridization solution was heated to 95°C for 5 minutes then cooled on ice for 5 minutes. This was then added to the tissue and incubated for 3 days at 45°C.
Post hybridization.
The hybridized tissue was then washed: 4 x 20 minutes at 45°C with prehybridization solution pre-warmed to 45°C, 1 X 30 minutes at room temperature with 2X SSC, 1 X 1 hour at 45°C in 2X SSC. 1 X 1 hour at 45°C with 0.1X SSC, and 4 X 15 minutes at room temperature with PBT. The tissue was then blocked for at least an hour with 1% BSA in PBT and then incubated over night with an anti-DIG antibody in 1% BSA in PBT solution (1:2000 dillution).
The tissue was then washed 3 X 5 minutes with PBT, 4 X 20 minutes with PBT and 2 X 5 minutes with alkaline phosphatase buffer (0.1M NaCl, 0.05M MgCl, 0.1M Tris pH 9.5, 0.01%
102 Tween 20, (levamisole – optional). 20ul of NBT/BCIP (Roche Cat.no. 11681451001) per 1ml of alkaline phosphatase buffer was then added until a signal became visible. The staining reaction was stopped with 2 X 5 minute washes with PBT. Tissue was mounted in 80% glycerol solution and observed under light microscope.
Production of antigen for antibody development.
Using the Abdesigner web site (http://helixweb.nih.gov/AbDesigner/), the amino acid sequence of the genes, CG7882, CG9509, CG15406, CG3285, CG3349, CG14292, 14069, CG13793, was analyzed for optimal immunizing peptides for antibody production. Based on these results, primers were developed for amplifying the desired peptide from a cDNA library (see Table 5 for primer list). RNA was isolated from accessory glands using the “RNeasy Mini Kit” (Cat.no. 74104 – QIAGEN). The “OneStep RT-PCR Kit” (Cat.no. 210210 - QIAGEN) was used to obtain the nucleotide sequence of the peptides directly from the RNA isolate. The primers used carried BamHI sites and were designed for subcloning the OneStep RT-PCR products into pQE-31. The initial cloning was done in DH5α cells. The plasmid was retransformed into SG13009(pREP4) cells and the expression protocol described in “The QAIexpressionist” (march 2001 fifth edition - QIAGEN) was followed to check for expression of the peptide. For the SDS-PAGE analysis, standard protocols were followed using 4-20%
gradient gels.
The peptides produced from the pQE-31 vector are tagged with 6xHis. This allows for easy purification using Ni-NTA following the native condition purification procedure described in the “The QAIexpressionist” (march 2001 fifth edition - QIAGEN).
103 Purification efficiency was tested by running the samples on a 4-20% gradient gel after which a western blot (Protein Blotting Guide – Bio-Rad, third edition) was performed using an anti-His antibody (Covalab mab 9001) or anti-RGS antibody (Cat.no.34698 - QIAGEN). Visualization was performed using a secondary antibody (Cat.no. S3721 - Promega) coupled with alkaline phosphatase and using the standard NBT-BCIP a colorimetric assay.
Concentration of the peptides was determined using the “Bio-Rad Protein Assay”, Microassay procedure. The samples were lyophilized and sent to Pocono Rabbit Farm &
Laboratory (PRF&L, PA 18325, USA) for injection in Rabbits using the Pocono Rabbit Farm &
Laboratory (PRF&L, PA 18325, USA).
Fly crosses and strains.
All crosses were done using standard genetic techniques. Fab71, iab-7Sz, iab-6,7IH, iab-5,6J82, and iab-4,5,6DB are described in (Mihaly, Barges et al. 2006). The lines iab-6∆5 iab-6∆6 and iab-64 are described in (Iampietro, Gummalla et al. 2010). The line Iab-7blt described in (Galloni et al., 1993); OR; the line Pc3 described in (Lewis, 1980); Pcl,Asx; Sw(Hw); the line Mcp1described in (Duncan, 1982); the line Zop6 described in (Lifschytz and Green, 1984); the line Abd-BD16 described in (Karch et al. 1985); Df(3L)0463 (CTCF) described in (Mohan, Bartkuhn et al. 2007); CTCFp306; the line Abd-BD18 described in (Hopmann et al. 1995); Df(3R)P9 described in (Lewis 1980); UAS-Rab4-GFP, UAS-Rab5-GFP, UAS-Rab7-GFP, UAS-Rab11-GFP, UAS-SARA-GFP, UAS-FYVE-GFP, UAS-Hrs-GFP, UAS-Uif-ecd-GFP, UAS-Uif-C-term-GFP (lines donated by Marcos Gonzales-Gaitan lab); the line iab-6∆5 was described as a deficiency without any phenotypic consequence. Following the LTR phenotype identified in this work the line was renamed iab-6cocu (reflecting that mates of these males fail to reject other suitors; “cocu” means “cuckold” in French). The BAC-AbdBGal4,w+, UAS-GFP/Cy line carrying
104 the Abd-B-Gal4 BAC reporter and a UAS-GFP marker on the second chromosome was created for this study by recombining a chromosome carrying the BAC and a UAS-GFP chromosome.
The BAC reporter chromosome cannot exist as a homozygote. The 4.4E transgenic lacZ reporter line is described in (Mihaly, Barges et al. 2006). The Gal4 expressing lines driven by a paired enhancer ((w-; prd-mf5.2,w+/CyO), (w-;prd-mf5.4,w+), (w-;prd-mf5.5,w+), (w-;prd-mf9.3,w+), (w
-;prd-mf9.7,w+)) were obtained from Makus Noll’s laboratory (Jiao, Daube et al. 2001). They were used in a cross with a UAS-AbdBm (Castelli-Gair, Greig et al. 1994) flies for the experiment in which we tested for the ability of Abd-Bm to transform main cells into secondary cells. Flies for the RNAi screen of the candidate genes were obtained from the VDRC (Vienna Drosophila Rnai Center) a list of which is provided in a table below.
Antibody, X-Gal and FM4-64 staining.
Antibody and X-Gal staining on embryos and dissected accessory glands was performed as described in (Hagstrom, Muller et al. 1996) and (Galloni, Gyurkovics et al. 1993) respectively, using a 20min fixation. The Abd-B primary antibody, obtained from the Developmental Studies Hybridoma Bank, was diluted 1:4. Goat-anti-mouse secondary antibody, coupled to Alexa Fluor 488/555 X (Invitrogen AG), used to reveal Abd-B localization was used at 1:500 dilution. Goat HRP coupled anti-mouse was obtained from Biorad and used at 1:1’500 dilution. Staining with FM4-64 dye was done by placing a drop of the dye onto a microscope slide and placing a freshly dissected gland into it. The glands were immediately covered with a cover slip and visualized using fluorescent microscope at 555nm.
105
C Sx/A new F SalI AAAAAGTCGACCAGCTCAACAGTCACACATAGACAG
F CS/A rec CATCGGACCACCCGCACTTG
106
R CS/A rec GCTAATGAGAGCGTTGAGAG
C-t mCh-fus F CCACCACCTGAACCTTAGCCTGAACATGGGTCACCATGCC
GCCAAGATGCACCAGATGGTGAGCAAGGGCGAGGA
C-t mCh-fus R ACTGCTAGTAGGTGGCAAAGCCCATGGCGCCAGCGCTGCT
GTTGTCCAAGGGTCATTACTTGTACAGCTCGTCCA
mCh F KpnI AAAAAGGTACCATGGTGAGCAAGGGCGAGGA
mCh R NotI AAAAAGCGGCCGCTTACTTGTACAGCTCGTCCA
mCh R NLS NotI AAAAAGCGGCCGCTTAGTGGAACGCGAAGAAGAACCCCTT
GTACAGCTCGTCCA
N-t mCh-rep F ACCCACCGCCCCGCACCCGCATCCGCATCCCCAGGCGCAT
CCCTCCCTGCCCGTCATGGTGAGCAAGGGCGAGGA
N-t2 mCh-rep R TGCTGCGGAGCGGGGAGGTGTTGCTGCTGTACGGCGACAA
GTGGCACAGGCGGAGTTCGCTATTACGCCAGCCCG
Table 1. List of primers used to perform the different BAC constructs described in material and methods section.
Primer table Creation of a specific secondary cell D5/DI-Gal4 drivers
D5 F AAAAAGGATCCCAGGAGCAATCCATCAAA
D5 R AAAAAGGATCCACAGCTCTGCTTTTTGCTGA
DI R AAAAAGGATCCGGCCGCCCAATGGATGTACA
Table 2. List of primers used to perform the different Gal4 driver constructs described in material and methods section.
107 Primer table Dissection and replacement of the cocu enhancer
F I GGCAGCACGAATAGTTTAGTTTATTTTAGCCATAGCTCAAG
AACGACAGCGAATACAAGCTTGGGCTGCAGG
R I GGTGAATAATTTTTATTGCCGTAAATCACTGTGTCAATTGT
GGTTGTAATCTCGCCCGGGGATCCTCTAGAG
F II TCCGACTCCAATACGAAATTAATTGGTTCCGATTGCAACTG
AGCGCAGTCGAATACAAGCTTGGGCTGCAGG
R II AGCATTTGTTTATCTGAAATTTTAATACGCTCCTTAATTTTT
ATGGGTTCCTCGCCCGGGGATCCTCTAGAG
F III CAAATTTTATGCTTTGTCACTGAAACGATTATGACGTCCGT
TGCTCGTCCGAATACAAGCTTGGGCTGCAGG
R III TTGGCAACAAAGTTGGATGCATTGTGGGTGGCAAAATATC
AAACAATGGCCTCGCCCGGGGATCCTCTAGAG
F rec mfKF GGCAGCACGAATAGTTTAGTTTATTTTAGCCATAGCTCAAG
AACGACAGCTTGCCACATTGTGTGTGGAC
F mf9 NcoI AAAAACCATGGTTGCCACATTGTGTGTGGAC
R mf9 EcoRI AAAAAGAATTCGCCCAGTTCTCGCAGTTCGA
F KF EcoRI AAAAAGAATTCGAATACAAGCTTGGGCTGCAGG
R KF SpeI AAAAAACTAGTCTCGCCCGGGGATCCTCTAGAG
Table 3. List of primers used to perform the different dissection and replacement constructs described in material and methods section.
108 Primer table FISH Abd-B locus probes
FISH1 AB F GAAACCAGCCCAACAATGAG
Primer table FISH 51C locus probes
FISH1 51C F ATGCAAGTAAAACAGTCGAG
Table 4. List of primers used to create FISH probes for the Abd-B and the 51C locus described in the materials and methods secton.
109 Primer table Antigen Expression
cg7882ant-F AAAAAGGATCCAATGGCAAAGCCAAAAGTCGG cg7882ant-R AAAAAGGATCCCTAATTGAGGGACCAGCTCCACT cg9509ant-F AAAAAGGATCCAAAGTCGGTCACGCCTCAAGG cg9509ant-R AAAAAGGATCCCTAATATCCTTTGCCTGTGCTCA cg3285ant-F AAAAAGGATCCACCGGAAACCCCACATCACCT cg3285ant-R AAAAAGGATCCCTAATCTCTGTAGTCGAAGGACT cg14069ant-F AAAAAGGATCCAAACTGGTCGGCTCAAATAGC cg14069ant-R AAAAAGGATCCCTAGGCCAACTTGCGACATCGTC cg15406ant-F AAAAAGGATCCAAGGTGGAAGCGCGAGGAGGA cg15406ant-R AAAAAGGATCCCTAATCGGACATTGAGAGGCCAT cg3349ant-F AAAAAGGATCCACGAGTTTGCCGCAAAGTATG cg3349ant-R AAAAAGGATCCCTATTCACCGTCCTTCGACAGCC cg13793ant-F AAAAAGGATCCATACTACATTCATCAGAGACC cg13793ant-R AAAAAGGATCCCTAGTTAACCTCCTCGGTGCGAT cg14292ant-F AAAAAGGATCCACTCTCCCAGCCTGACTTTCC cg14292ant-R AAAAAGGATCCCTAGGCAGCAGCTGCTCCTGTGG
Table 5. List of primers used to perform the different antigen expression constructs described in material and methods section.
110
111
Table 6. List of fly lines with the corresponding transformant ID obtained from the VDRC used in an RNAi experiment
described in material and methods section.
112 Primer table In Situ Hybridization
CG7882 S CATTAACGTTCTCAGAGGCG
CG7882 AS CAAACAGTTCTGCTCCGATG
CG9509 S ATCCGTTCCAGCAACACCTG
CG9509 AS ACGCACAGCAAATGGGAGTC
CG15406 S CATCTTCCAGTGGCAGTACC
CG15406 AS GCACAGAACCACATGGTAGC
CG3285 S CTATTGGGACGCAGGATTCG
CG3285 AS CTGTACTGCTTAGGATCACC
CG3349 S GCCAAGTTAGCAGTCAAGTC
CG3349 AS TTTGATGCCATTCTTCTCCG
CG14292 S TCGCAATTGTTCTACTGGCG
CG14292 AS AAACGGGCCTGCCACTCGTC
CG14069 AS CTAGGCCAACTTGCGACATC
CG14069 S CAATTGCCATCGGATTGTTA
CG13793 S ATTGGGAAAAACCCACGGAC
CG13793 AS GGTGGAGCTACGAAACGGAG
B gal S CCAGGCGTTAGGGTCAATGC
B gal AS GTTAACCGTCACGAGCATCA
AbdBr S CCATACAATTAGCGCCACTG
AbdBrAS TGACCCATGTTCAGGCTAAG
AbdBm S CAGCAGCAGCAACATGCAGT
AbdBm AS GATAATCCACCAGAGGCTCC
Table 7. List of primers used to make probes for in situ hybridization.
113
CG17752 3,78 0,00 378334,00 transmembrane transsporter
Malpighian
Tubules Yes
CG33775 1,93 0,00 193250,00 unknown ubiquitously
expressed No
CG15406 1,52 0,00 152422,00 transmembrane transsporter
Malpighian
Tubules No
CG3285 1,28 0,00 127926,00 transmembrane transsporter
Malpighian
Tubules Yes
CG2196 1,28 0,00 127926,00 sodium:iodide symporter activity
Malpighian
Tubules No
CG18211 1,14 0,00 114317,00 serine-type endopeptidase
activity Midgut Yes
CG6298 0,98 0,00 97985,70 serine-type endopeptidase
activity Midgut No
CG8197 0,79 0,00 78932,90 unknown Testis No
CG14069 0,68 0,00 68045,60 cytokine activity Testis Yes
CG31034 0,68 0,00 68045,60 serine-type endopeptidase
activity unknown Yes
CG18444 0,65 0,00 65323,80 serine-type endopeptidase
activity Midgut Yes
CG12374 0,65 0,00 65323,80 metallocarboxypeptidase
activity Midgut Yes
CG13656 0,65 0,00 65323,80 unknown Malpighian
Tubules Yes
CG12809 0,63 0,00 62602,00 zinc ion binding; nucleic
acid binding Brain No
CG6733 0,63 0,00 62602,00 aminoacylase activity Malpighian
Tubules No
CG10152 0,63 0,00 62602,00 unknown Brain Yes
CG31272 0,54 0,00 54436,50 lipase activity; transporter activity
Malpighian
Tubules No
CG33783 34,54 0,03 1043,24 unknown unknown No
CG33784 18,97 0,03 573,00 unknown Eye yes
CG33631 5,31 0,03 160,31 unknown ubiquitously
expressed yes
CR11700 13,96 0,20 70,29 unknown unknown unknown
CG11598 941,23 14,24 66,11 lipase activity; triglyceride lipase activity
CG9036 1,74 0,03 52,61 structural constituent of
chitin-based cuticle; Testis yes
114
structural constituent of chitin-based larval cuticle CG15902 1,52 0,03 46,04 glucuronosyltransferase
activity Midgut yes
CG14376 12,49 0,30 41,93 ligand-gated ion channel activity
ubiquitously
expressed yes
CG13538 1,36 0,03 41,10 unknown Testis No
CG7874 24,63 0,63 39,16 extracellular matrix structural constituent
Malpighian
Tubules yes
CG8687 5,91 0,17 35,68 electron carrier activity Crop yes CG9294 2,29 0,07 34,53 serine-type endopeptidase
activity
ubiquitously
expressed No
CG13793 40,53 1,26 32,21 neurotransmitter
transporter activity Heart No
CG7882 1,99 0,07 30,01 glucose transmembrane transporter activity
CG14681 22,26 0,79 28,02 structural molecule activity unknown yes CG9509 1,74 0,07 26,31 choline dehydrogenase
activity
Malpighian
Tubules No
CG13309 0,87 0,03 26,31 chitin binding Malpighian
Tubules yes
CG5106 4,27 0,17 25,81 unknown Testis yes
CG1089 2,53 0,10 25,48 carboxylesterase activity Midgut no
CG42235 2,53 0,10 25,48
CG5361 1,47 0,07 22,20 alkaline phosphatase activity CG1112 2,18 0,10 21,92 carboxylesterase activity ubiquitously
expressed no
CG6467 0,71 0,03 21,37 serine-type endopeptidase
activity Midgut yes
CG2187 0,60 0,03 18,09 sodium:iodide symporter activity
Malpighian
Tubules No
115
CG14292 2,40 0,13 18,09 unknown Malpighian
Tubules Yes
CG31198 0,57 0,03 17,26 metallopeptidase activity;
zinc ion binding Midgut Yes
CG33630 0,54 0,03 16,44 unknown ubiquitously
expressed yes
CG3048 11,30 0,76 14,83 protein binding Heart No
CG5207 7,57 0,53 14,28 unknown Testis yes
CG15614 1,22 0,10 12,33 G-protein coupled receptor activity
CG11205 12,25 1,09 11,21 deoxyribodipyrimidine photo-lyase activity
CG7171 0,63 0,07 9,45 urate oxidase activity Malpighian
Tubules No
CG10118 0,68 0,10 6,85 tyrosine 3-monooxygenase
activity Brain No
CG10764 0,65 0,10 6,58 serine-type endopeptidase
activity Testis yes Table 1. Showing the fold decrease of the 73 candidate genes as well as their predicted molecular function and the tissue of maximum expression. Final 8 candidates are marked in yellow.
116
117
118
119
Table 2. Five day egg laying data from the RNAi experiment for the candidate genes available from the VDRC.
Descriptive data on the secondary cell morphology also included. Final 8 candidates are marked in yellow.
120
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