Functional analysis of the nuage, a unique germline organelle, in drosophila melanogaster 2

For high fidelity amplification, either PfuUltra Stratagene or Pfx Invitrogen polymerase was used in accordance to the manufacturers’ instructions.. The cells were used directly for tran

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2 Materials and Methods

2.1 Molecular work

2.1.1 Recombinant DNA methods

2.1.1.1 Strains and culture conditions

Escherichia coli strains DH5α or XL1-Blue was used for all recombinant DNA

procedures unless otherwise stated For the use of restriction endonucleases that are

sensitive to dam methylation, a dam negative strain JM110 was used For TOPO cloning, TOP10 E coli (Invitrogen) was used For protein expression in E coli,

pENTR/D-BL21(DE3) was employed All bacterial strains were cultured in Luria-Bertani [LB; 1% (w/v) bacto-tryptone, 0.5% (w/v) yeast extract, 1% (w/v) sodium chloride (NaCl)] broth

or grown on agar plates at 37°C For drug resistance selection, the culture media was supplemented with the antibiotics ampicillin (100 μg/ml), kanamycin (50 μg/ml), or chloroamphenicol (37 μg/ml)

2.1.1.2 Bacterial glycerol stocks

A fresh colony was cultured in LB, supplemented with the respective antibiotics, by shaking at 250 revolutions per min (rpm) at 37°C until the optical density (OD600) reached 0.8 Glycerol was added to a final concentration of 12% (v/v) on ice and the glycerol stock was stored at -80°C

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2.1.1.3 Plasmid DNA preparation

For small-scale and large-scale plasmid DNA preparation, Geneaid High Speed Mini Kit and Geneaid Plasmid Maxi Kit were used in accordance to the manufacturer’s instructions, respectively DNA concentration was determined using NanoDrop ND-1000 Spectrophotometer (BioFrontier Technology)

2.1.1.4 Polymerase Chain Reaction (PCR)

Standard PCR and colony PCR were performed in the presence of 50 ng template DNA,

200 μM deoxynucleotide triphosphate (dNTP), 500 μM of each primer (forward and reverse), and 0.4 units (U) Taq polymerase (iDNA Biotechnology) with the cycling conditions: 1 cycle of 94°C for 2 min, 25-33 cycles of 94°C for 30 sec/55-62°C for 30 sec/72°C for 1 min per kb, 72°C for 10 min For high fidelity amplification, either PfuUltra (Stratagene) or Pfx (Invitrogen) polymerase was used in accordance to the manufacturers’ instructions All primer sequences can be found in Table IV

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94°C for 2 min/50°C for 30 sec/60°C for 4 min, 60°C for 10min The reactions were then analysed on 3730x1 DNA Analyser (Applied Biosystems)

2.1.2 Bacterial transformation

2.1.2.1 Preparation of heat-shock competent cells

E coli cells were cultured in SOB [2% (w/v) bacto-tryptone, 0.5% (w/v) yeast extract, 10

mM NaCl, 2.5 mM potassium chloride (KCl), 10 mM magnesium chloride (MgCl2), 10

mM magnesium sulphate (MgSO4)] at 37°C until the OD600 reached 0.6 The cells were then chilled on ice for 10 min and harvested by spinning at 3000 rpm at 4°C The pellet was resuspended in TB media [10 mM 1,4-Piperazinediethanesulfonic acid, Piperazine-1,4-bis(2-ethanesulfonic acid), Piperazine-N,N′-bis(2-ethanesulfonic acid (PIPES), 55

mM manganese chloride (MnCl2), 15 mM calcium chloride (CaCl2), 250 mM KCl] and incubated on ice for 10 min The cell suspension was centrifuged and fresh TB was added

to the bacterial pellet The cells were used directly for transformation or alternatively, stored for long-term in 7% (v/v) dimethyl sulfoxide (DMSO) at -80°C

2.1.2.2 Preparation of electrocompetent cells

E coli cells were cultured in LB broth at 37°C until the OD600 reached 0.5-0.7 The cells were then chilled on ice for 10-15 min and harvested by spinning at 4200 rpm at 4°C Following two resuspensions in ice-cold water, the cells were ready for electroporation Electrocompetent cells were stored for long-term in 10% (v/v) glycerol at -80°C

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2.1.2.3 Transformation

For heat-shock transformation, competent cells were mixed with an appropriate volume

of DNA and incubated on ice for 30 min The DNA/cell mixture was then heat-shocked

at 42°C for 60 sec and immediately placed on ice For electroporation, competent cells were mixed with an appropriate volume of DNA and transferred to a pre-chilled electrocuvette on ice The DNA/cell mix was pulsed once at 2.5 kilovolts (kV) and 25 microfarad (μF), and immediately placed on ice Following either transformation, 1 ml of

LB was added to each mixture and mixed gently before incubating the culture at 37°C, with shaking at 250 rpm for 1 hr to allow for plasmid expression The cells were then plated on LB agar supplemented with the respective antibiotics, depending on the plasmids

2.1.3 Cloning strategies and constructs

2.1.3.1 Conventional cloning

Restriction digests of vector DNA and PCR fragments were performed in accordance to the manufacturers’ instructions To dephosphorylate the 5’- end of the digested vector DNA, 1 U of calf intestinal phosphatase (Roche) was added to the restriction digest and incubated at 37°C for 60 min To fill the 3’- end of staggered DNA, 1 U of Klenow enzyme (Roche) and 1 U of polynucleotide kinase (Roche) was added to the digested DNA and incubated at 37°C for 20 min, in the presence of 20 pmol adenosine triphosphate (ATP) and 250 μM dNTP Ligation was set up with a vector:insert molar ratio of 3:1, in the presence of 3 U T4 DNA ligase (Roche) Sticky-end and blunt-end

II, Invitrogen), followed by a site-specific recombination step to swap the DNA from

pENTR™/D-TOPO® into the destination vectors (Appendix III, The Drosophila

Gateway™ Vector Collection,

For TOPO cloning, PCR product with a flanking CACC sequence at the 5’- end and a blunt 3’- end was mixed with pENTR™/D-TOPO® in a molar ratio of 2:1, and incubated

at room temperature for 30 min Half the ligation mixture was then transformed into

OneShot® TOP10 chemically competent E coli (Invitrogen) in accordance to the

manufacturer’s instructions

For site-specific recombination, equal volumes of pENTR™/D-TOPO® harbouring the gene-of-interest or DNA fragment and the Gateway® destination vectors were mixed, in the presence of LR Clonase™ II enzyme mix (Invitrogen) The DNA/enzyme mix was incubated at 25°C for 2 hr to promote recombination The reaction was subsequently terminated by adding Proteinase K and incubating at 37°C for 20 min One-half of the reaction mix was used for transformation

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2.1.3.3 TA cloning

PCR products that were amplified using Taq polymerase were used directly for TA cloning For blunt-ended PCR products, A-tailing was performed by incubating the purified DNA duplex with 0.2 mM dATP and 5 U Taq polymerase at 70°C for 30 min

For TA cloning, pGEM®-T Easy vector (Promega) and insert DNA were mixed in the molar ratio 1:3 and incubated for 60 min at room temperature or overnight at 4°C (up to

16 hr) One-half of the ligation mix was then used for transformation Recombinant clones were identified using the blue-white selection, where the presence of an insert in

pGEM®-T Easy vector disrupts the ß-galactosidase gene and produces white bacterial

colonies 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-Gal) and isopropyl thiogalactopyranoside (IPTG) were added as substrates, and to a final concentration of 0.5 mM and 80 μg/ml, respectively

β-D-1-2.1.4 Single-fly PCR

2.1.4.1 Preparation of fly genomic DNA

D melanogaster genomic DNA was prepared by mashing a single fly in 50 μl squishing

buffer [10 mM Tris-Cl pH 8.2, 1 mM ethylenediaminetetraacetic acid (EDTA), 25 mM

NaCl, and 200 pg/ml freshly diluted Proteinase K (Sigma)] with a pipette tip (Gloor and

Engels, 1992; Gloor et al., 1993) The squished fly was subsequently incubated at 37°C for 20-30 min, followed by a heat-inactivation step at 65°C for 10 min Single fly genomic DNA preps were stored at 4°C or -20°C

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2.1.4.2 Genomic DNA PCR

Genomic DNA PCR was performed as described in 2.1.1.4 1 μl of genomic DNA was used per PCR

2.1.5 Total RNA extraction

Ovaries were dissected in cold Grace’s medium and transferred to TRIzol reagent (Invitrogen) on ice Total RNA was extracted from the ovaries in accordance to the manufacturer’s instructions Extracted total RNA was reconstituted to a final concentration of approximately 2-3 μg/μl and stored at -80°C

2.1.6 Poly A + RNA purification

Total RNA extracted from at least 50 ovaries was used for poly A+ RNA purification using the Oligotex mRNA kit (QIAgen) in accordance to the manufacturer’s instructions

2.1.7 DNase treatment

DNase treatment was performed by incubating total RNA with DNaseI (Roche) at room temperature for up to 15 min 1 U of DNaseI was used per μg total RNA Following treatment, DNaseI was heat-inactivated by adding EDTA pH 8.0 to a final concentration

of 2.5 mM and incubating the samples at 65°C for 10 min

2.1.8 Reverse transcription (RT)

1 µg of DNase-treated total RNA was reverse-transcribed using oligo(dT)15 and Superscript III reverse transcriptase (Invitrogen) A mock reaction without reverse

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transcriptase was also prepared for each RNA sample The newly-synthesized complementary DNAs (cDNAs), following treatment with RNaseH (Stratagene), were first normalised and checked for genomic DNA contamination by performing PCR with

actin5C (act5C) or alcohol dehydrogenase (adh) primers

2.1.9 Semi-quantitative and quantitative PCR

Semi-quantitative PCR was performed as described in 2.1.1.4 using 1 µl per cDNA sample/reaction Quantitative PCR was similarly carried out, except with a change to the use of Bio-Rad MyQ™ Single-Colour Real-Time PCR Detection System, in the presence

of iQ™ SYBR® Green Supermix (Bio-Rad) Control experiments measuring the change

in CT with template dilution were performed on the target genes and the control act5C All the results were normalised with respect to act5C P-values were measured using one-

tailed student T-test All primer sequences can be found in Table IV

2.1.10 Poly(A) tail test (PAT)

2.1.10.1 Rapid Amplification of cDNA Ends-PAT (RACE-PAT)

1-3 µg of DNase-treated ovarian total RNA was reverse-transcribed using oligo(dT)anchorand Avian myeloblastosis virus (AMV) Reverse Transcriptase (Promega, (Salles et al., 1999) A mock reaction without reverse transcriptase was prepared for each RNA sample The newly synthesized cDNAs were checked for genomic DNA contamination by PCR

with act5C primers PCR was subsequently performed using 1 µl diluted or neat cDNA

sample/reaction as described in 2.1.1.4 For analyses of retroelement de-repression,

primer sets corresponding to act5C and HeT-A untranslated regions (UTRs), coding

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sequence (CDS), and poly(A) regions were used All primer sequences can be found in Table IV

2.1.10.2 Ligation-mediated PAT (LM-PAT)

3 µg of total RNA from ovaries from each sample was treated with DNaseI as described

in 2.1.7 LM-PAT assay was performed as described in (Salles et al., 1999) Amplified products were visualised on 3% (w/v) agarose/1x Tris Boric EDTA gel All primer sequences can be found in 2.6

2.2.1 Fly husbandry and stocks

D melanogaster were cultured on standard cornmeal-agar medium at 25°C For ovary

staining, y w was used as a wild-type control unless otherwise stated FM6, sco/CyO and

TM3/TM6B were used as first, second, and third chromosome balancers, respectively

Mutant alleles and allelic combinations used were vas PH165 (Styhler et al., 1998),

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krimp f06583 (Bloomington Drosophila Stock Center), krimp f06583 /Df(2R)Exel6063

(Bloomington Drosophila Stock Center), aub N11/HN2(Schupbach and Wieschaus, 1991;

Wilson et al., 1996), mael M391 /Df(3L)79E-F (Clegg et al., 1997; Hartenstein and Jan,

1992), spn-E 616/hls3987 (Gillespie and Berg, 1995; Gonzalez-Reyes et al., 1997),

dcr-2 L811fsX (Hatfield et al., 2005), armi KG04664 (Bloomington Drosophila Stock Center), b53;

T3 (an insertion mutant for dcp1 that harbours a rescue transgene for a neighbouring

gene, CG5602; (Lin et al., 2006), twin KG00877 /Df(3R)crb-F89-4 (Bloomington Drosophila

Stock Center), ski3 f03251 /Df(2R)Np5 (Bloomington Drosophila Stock Center), and pcm ∆1

(Appendix I) As homozygous dcr-1 Q1147x is lethal, mitotic clones were generated by

subjecting 2-day old female flies [hsFLP/eyFLP;FRT82BUbi- green fluorescence protein (GFP)/FRT82Bdcr-1 Q1147x] to heat-shock at 37°C for 1.5 hr The treated flies were then aged for 7 to 10 days prior to immunostaining Me31B-GFP and Protein disulfide isomerase (PDI)-GFP fly lines were obtained from Carnegie Protein Trap Library

(Buszczak et al., 2007) Flies carrying the transgene, UASp-aub-GFP (Harris and Macdonald, 2001), UASp-dcp1-Haemagglutinin (HA) (Lin et al., 2006), UASp-krimp- Venus[Yellow fluorescence protein (YFP)] or UASp-krimp(variants)-MYC were crossed

to flies harbouring the nosgal4VP16 transgene to drive the expression of tagged proteins

in the female germline (Van Doren et al., 1998) For ms2/MS2 Coat Protein (MCP) labeling of the retroelement transcripts, flies carrying UASp-hs-HeT-A-(ms2) 6, UASp-hs-

I-element-(ms2)6 or the control UASp-hs-nanos(nos)-(ms2) 6 were crossed to those expressing MCP-GFP (Forrest and Gavis, 2003) and the F1 progenies were subjected to a heat-shock treatment to induce gene expression (see 2.5)

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2.2.2 Generation and clean-up of mutant alleles

pcm loss-of-function allele was generated by standard imprecise excision (Bachmann and

Knust, 2008; O'Connor and Chia, 2002) of the P-element insertion EP1526 (Bloomington

Stock Center), a homozygous viable DNA element that was inserted 584 nt downstream

of the polyadenylation site EP1526 was mobilised by crossing virgin females that carried this insertion to males that possessed the ∆2-3 transposae (Bloomington Stock Center)

More than 100 independent excision lines were screened for the deletion of pcm CDS

using single-fly PCR (described in 2.1.4.2 and Appendix I)

The original krimp f06583 and ski3 f03251mutant lines from the Stock Center exhibited male sterility and lethality, respectively due to background mutations Both fly stocks were

cleaned up by backcrossing to y w prior to the use of these alleles for all the experiments

described

2.2.3 Generation of transgenic flies by microinjection

1 hr old y w embryos were collected on cornmeal-agar plates and dechlorionated in 50%

(v/v) chlorox for 2 min Dechlorionated embryos were washed extensively with deionised water, aligned on a nitrocellulose membrane and then adhered onto a coverslip The embryos were dried in silica gel for approximately 12-15 min and covered with a thin layer of halocarbon oil Plasmid DNA and helper DNA were injected manually under the microscope at a concentration of 100 ng/μl and the embryos were allowed to hatch in a

wet chamber The emerged flies were crossed to sco/CyO or TM3/TM6B balancer flies

Transgenic flies were identified by the orange eye colour in the F1 progenies

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Transgenes that were microinjected include krimp full length (FL)-Venus,

UASp-krimp N-terminus (NT)-MYC, UASp-UASp-krimp C-terminus (CT)-MYC, UASp-ago3FL-HA,

pCaSpeR-hs-HeT-A-(ms2) 6 , and pCaSpeR-hs-I-element-(ms2) 6 For the construction of

UASp-krimp(variants)-Venus/MYC and UASp-ago3FL-HA, expressed sequence tag

(EST) clones RE66405 and LD17152 were used as templates, respectively The PCR products were cloned into pENTR™/D-TOPO® (Invitrogen) and recombined into the destination vectors pPVW, pPMW, and pPHW using Gateway® technology (see 2.1.3.2)

The construction of pCaSpeR-hs-HeT-A-(ms2) 6 and pCaSpeR-hs-I-element-(ms2) 6 are described in 2.5.1 All primer sequences can be found in Table IV

2.3 Immunohistochemistry and Microscopy

2.3.1 Antibody staining of fixed ovaries

Ovaries were dissected in Grace’s medium (BioWhittaker) and immediately fixed for 5 min at room temperature in the fixative solution (2 parts of Grace’s medium to 1 part of 16% paraformaldehyde, electron microscopy grade) After several washes with PBX {PBS [10 mM sodium phosphate monobasic (NaH2PO4)/sodium phosphate dibasic (Na2HPO4) pH 7.4, 175 mM NaCl] containing 0.1% Triton X-100}, the fixed ovaries were pre-absorbed in PBX containing 5% normal goat serum (The Jackson Laboratory) for at least 30 min at room temperature The ovaries were then rinsed twice with PBX and incubated with the primary antibodies diluted in PBX containing 0.5% (w/v) bovine serum albumin (BSA, Sigma) for 4 hr at room temperature or overnight at 4°C After several washes in PBX, the ovaries were incubated in secondary antibodies diluted in