Suberites domuncula, NmeGp1Sd. The purpose of this study was to investigate the properties of the sponge Group I Nme gene and protein, and compare it to its human homolog in order to elucidate the evolution of the structure and function of Nme.Nucleoside diphosphate kinases NDPK are evolutionarily conserved enzymes present in Bacteria, Archaea and Eukarya, with human Nme1 the most studied representative of the family and the first identified metastasis suppressor. Sponges (Porifera) are simple metazoans without tissues, closest to the common ancestor of all animals. They changed little during evolution and probably provide the best insight into the metazoan ancestor's genomic features. Recent studies show that sponges have a wide repertoire of genes many of which are involved in diseases in more complex metazoans. The original function of those genes and the way it has evolved in the animal lineage is largely unknown. Here we report new results on the metastasis suppressor gene/protein homolog from the marine sponge We found that sponge genes coding for Group I Nme protein are intron-rich. Furthermore, we discovered that the sponge NmeGp1Sd protein has a similar level of kinase activity as its human homolog Nme1, does not cleave negatively supercoiled DNA and shows nonspecific DNA-binding activity. The sponge NmeGp1Sd forms a hexamer, like human Nme1, and all other eukaryotic Nme proteins. NmeGp1Sd interacts with human Nme1 in human cells and exhibits the same subcellular localization. Stable clones expressing sponge NmeGp1Sd inhibited the migratory potential of CAL 27 cells, as already reported for human Nme1, which suggests that Nme's function in migratory processes was engaged long before the composition of true tissues.This study suggests that the ancestor of all animals possessed a NmeGp1 protein with properties and functions similar to evolutionarily recent versions of the protein, even before the appearance of true tissues and the origin of tumors and metastasis. The Nme family, initially called nucleoside diphosphate kinases (NDPK) or Nm23, are evolutionarily conserved proteins present in all three domains of life: Bacteria, Archaea and Eukarya . VertebrNme1 and Nme2 arose by cis-duplication  and Leucetta chagosensis [GenBank:GO094744 and GenBank:GO094741]. Phylogenetic analysis shows that distinct sponge proteins from the same species clearly group together, indicative of independent duplications  Figure . Bindingmyc promoter. It binds to the NHE sequence of the c-myc promoter cloned into pUC19 which yields mostly open circle (nicked circular) plasmid . The amplified fragment was separated on a 0.8% agarose gel, purified and sequenced. The reverse specific primer 5'- CATGGCAACCATCTTGAAGCC -3' was used in conjunction with the vector specific primer T3 to amplify the 5' upstream region from translation start of the nmeGp1Sd gene on the genomic library from S. domuncula [For genomic DNA preparation, the specimens of omuncula .Nucleotide sequences were stored and analyzed using Lasergene . Multiple sequence alignments (MSA) were performed with CLUSTALX . The exaNmeGp1Sd gene from the sponge Amphimedon queenslandica was identified in the genome (contig13509) available at http://spongezome.metazome.net/cgi-bin/gbrowse/amphimedon/[http://www.ncbi.nlm.nih.gov). NmeGp1 sequences from sponges Leucetta and Sycon were identified by blastn with nmeGp1Sd sequence against NCBI's dbEST limited to Porifera. Hamming clustering method for TATA signal prediction in eukaryotic genes was used for identifying TATA-box http://www.itb.cnr.it/sun/webgene/[http://www.cbrc.jp/research/db/TFSEARCH.html and TRANSFAC database [The homolog of the phimedon/. Homolog/webgene/. TFSEARCdatabase , with a NmeGp1Sd gene to perform PCR reactions on the S. domuncula cDNA library. The PCR generated cDNA fragment was cloned into pBluescript vector (Stratagene). The cDNA for NmeGp1Sd was recloned into pET15b (Novagen) using PCR into BamHI (5'-TTCCCGGATCCAAAAAGTTACTCATAGATC-3') and NdeI (5'-GCTTTATACATATGACAACCGAGCGTACC-3') restrictions sites of pET15b expression vector downstream from the thrombin cleavage site. For colocalization assay, the full-length coding sequence NmeGp1Sd was cloned in frame into pEGFPN1 and pDsRedN1 expression vectors . The fragment of 541 bp was amplified using 5'-GCTTTCTCGAGATGACAACCGAGCGTACC-3' and 5'-GTAGACGGATCCTTCTCATAGATCCATTGCTG-3' primers. The PCR product was digested with XhoI and BamHI, and ligated with the XhoI/BamHI sites of pEGFP-N1 and pDsRedN1 expression vectors. The pEGFPN1-Nme1 was a kind donation of Dr. Marie-Lise Lacombe, Faculty of Medicine St. Antoine, Paris, France. NmeGp1Sd cDNA was amplified using a forward primer containing the FLAG sequence (F: 5'-GTCTAGGGATCCACGAGATGGACTACAAGGACGACGACGATAAGATGACAACCGAGCGT-3') and the reverse primer (R: 5'-CTAGACGAATTCTTATTACTCATAGATCCATTGCTGTTCTGTGGG-3'). The PCR product was cloned into the BamHI/EcoRI sites of eukaryotic expression vector pcDNA3 (Invitrogen).We used the same set of primers as for Escherichia coli strain BL21 tagged with six histidine residues at the N-terminus and purified to homogeneity from bacterial lysates using nickel affinity chromatography. E. coli strain BL21 harboring the plasmid construct was grown to 0.6 at OD600 and induced with 0.8 mM IPTG for 3 hours at 37°C. Cells were incubated 30 min on ice in lysis buffer  and sonicated 8 × 30 sec (50% of the full power). After centrifugation (12000 rpm) for 25 min at 4°C, the supernatant was applied onto nickel-charged agarose column (Qiagen). Histidine tagged proteins bound to nickel-affinity resins were eluted with 500 mM imidazole. NmeGp1Sd protein was applied to a PD-10 desalting gel filtration column  equilibrated with 25 mL of Nm23 buffer [2, 0.1 mM EDTA, 2 mM DTT and 0.1 M KCl) and then eluted with 3.5 mL of Nm23 buffer. The polyhistidine tag was released from the fusion protein by cleaving with thrombin (Sigma) (2 U/40 μg of fusion protein) for 16 hours at room temperature. The human Nme1 protein was a kind donation of Dr. Ioan Lascu .NmeGp1Sd was overproduced in 3 buffer   and eluted with Nm23 buffer  at a floin vitro as described [2. After 15 min of incubation at 37°C, 4 μL of 40 mM Tris-acetate (pH 7.5) containing 50% glycerol and 0.01% bromophenol blue was added and the products were immediately subjected to gel electrophoresis at 3 V/cm in 0.6% agarose for 3 hours using 40 mM Tris-acetate (pH 7.5) and 1 mM EDTA as running buffer.The DNA-binding activity of the NmeGp1Sd protein was assayed escribed . Reactio2, 1 mM phosphoenolpyruvate, 0.1 mg/mL NADH, 1 mM ATP, 0.2 mM dTDP, 2 U/mL of pyruvate kinase, 2.5 U/mL of lactate dehydrogenase, and 1 mg/mL bovine serum albumin. All reagents and enzymes were purchased from Sigma. Reactions were initiated by the addition of 50 ng of NmeGp1Sd enzyme and activity was monitored in an Ultrospec® 2100 pro  measuring the decrease in absorbance at 340 nm. The control reactions omitting NmeGp1Sd produced minor rates but were subtracted from the NmeGp1Sd values. The same reaction was performed with human Nme1 enzyme.Enzyme-coupled Reaction-NDPK activity was measured using a coupled pyruvate kinase-lactate dehydrogenase assay ,59. Fivec-myc promoter (5'-GATCCCCAGTCTCCTCCCCACCTTCCCCACCCTCCCCACCCTCCCCATAAGCGAATT-3') insert into pUC19 plasmid. Recombinant plasmid pUC19-NHE (500 ng) was incubated with 500 ng of NmeGp1Sd protein. The reaction was assembled in 50 mM KCl, 10 mM MgCl2, 50 mM Tris HCl pH 7.5, 0.5 mM DTT, 30 μg/mL BSA and incubated 30 min at 20°C. The reaction was terminated with 1% SDS, 10 mM EDTA and proteinase K treatment (200 μg/mL) for 30 min at 55°C. Topoisomerase I (Gibco BRL), Nme1 and Nme2 proteins were used as controls and were processed in the same conditions. Samples were analyzed in 1% agarose gel in TAE buffer and than stained with 0.5 μg/mL ethidium bromide for 30 min.We cloned the double stranded 57-bp NHE (nuclease hypersensitive element) of the 2 at 37°C.Human head and neck tumor cell line HEp-2 (squamous cell carcinoma of the larynx) was obtained from ATCC, while CAL 27  were a kind donation of Dr. Jeannine Gioanni, Centre Antoine Lacasagne, Nice, France. Both cell lines were cultured in Dulbecco's modified Eagle medium  supplemented with 10% fetal bovine serum , 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin in humidified chamber with 5% CO4 HEp-2 cells were seeded on eight-well culture slides  to obtain 60% confluence. Each chamber was transfected with 1 μg of plasmid DNA: pEGFPN1-Nme1, pEGFPN1-NmeGp1Sd, or cotransfected with pDsRedN1-NmeGp1Sd and pEGFPN1-Nme1 (0.5 μg of each construct) using Lipofectamine reagent (Invitrogen) according to the manufacturer's instructions. Forty eight hours post transfection the cells were washed with PBS , fixed in 4% formaldehyde, and mounted in mounting medium (DAKO) supplemented with 1 μg/mL 4,6-diamino-2-phenylindole (DAPI) (Sigma) for nuclear staining.Twenty four hours before transfection 3 × 10Fluorescent images were obtained by Leica TCS SP2 AOBS laser scanning confocal microscopy equipped with HCX PL APO λ-Blue 63 × 1.4 objective. GFP was excited by 488 nm laser line, DsRed using 543 nm, and DAPI using 405 nm laser line.Stably transfected cell lines were prepared as described previously . In brie6 CAL 27 cells stably transfected with "empty" vector (K0), as well as CAL 27 cells stably expressing FLAG/NmeGp1Sd (S1 and S2) and FLAG/Nme1 expressing clones (K1 and K2) were seeded on 6-well plates overnight. Immunoprecipitation of FLAG/Nme1 was performed using anti-FLAG M2 affinity gel (Sigma) according to manufacturer's instructions.For detection of Nme1/NmeGp1Sd complex formation 1 × 10The proteins were separated by SDS-PAGE and electrotransfered to an Imobilon-PSQ membrane (Milipore). The membranes were incubated with anti-Nme1 antibody (Beckton-Dickinson) or with anti-FLAG M2 antibody (Sigma) and Nme1 antibody for detection of complex formation. Protein bands were visualized using Chemiluminiscence blotting substrate-POD (Roche) on Lumi-Film-Chemiluminiscent Detection Film (Roche).5 cells  and CAL 27 constitutively expressing NmeGp1Sd (S1 and S2)) overnight. After attachment the cells were starved in serum free medium for 24 hours at 37°C in a humidified chamber. After starvation the cell suspension (4 × 105 cells) was added to the upper chamber of Cell Culture Inserts (Beckton-Dickinson) and allowed to settle down for 10 min. DMEM supplemented with 10% FBS served as a chemoattractant and was added to the lower chamber. Cells were allowed to migrate for 20 hours. Nonmigratory cells were detached with cotton swabs, membranes were fixed in 4% formaldehyde, stained with 0.1% crystal violet, cut out from the inserts, mounted in mounting medium (DAKO) and analyzed by light microscopy. The migrated cells were analyzed at 400 × magnitude. Three images per membrane were acquired and analyzed in ImageJ program http://rsbweb.nih.gov/ij/index.html which measured the area of the membrane covered with migrated cells.Boyden chamber assay was done as described before . BrieflyDP carried out the molecular genetic studies, biochemical assays and the sequence analysis. MHB and RB carried out colocalization, immunoprecipitation and migration experiments. MD carried out stable clone preparation. AM participated in the biochemical assays and MH performed phylogenetic analysis. HĆ conceived and oversaw the project, participated in the sequence analysis and molecular genetic studies. HĆ, MHB, DP and MH wrote the manuscript. All authors read and approved the final manuscript.Promoter regions. The structure of NmeGp1 promoter regions from sponges S. domuncula and A. queenslandica. The most plausible putative binding sites for transcription factors identified by TFSEARCH are marked. Motifs shared with human Nme1 promoter region are boxed. Arrows denote the orientation of motifs. TSS - transcription start site.Click here for fileMaximum parsimony phylogenetic tree of Group I Nme members. Bootstrap values inferred from 1000 replicates are shown next to the branches (maximum parsimony/neighbour joining support). Accession numbers of sequences used are given in brackets after species names.Click here for fileMultiple sequence alignment of NmeGp1 proteins. Human (Nme1 and Nme2), choanoflagellate Monosiga brevicollis (NmeGp1Mb) and sponges Suberites domuncula (NmeGp1Sd), Amphimedon queenslandica (NmeGp1Aq), Sycon raphanus (NmeGp1SrA and NmeGp1SrB) and Leucetta chagosensis (NmeGp1LcA and NmeGp1LcB) proteins were aligned. Enzyme active site amino acids and amino acids necessary for the association of subunits on the hexamer are conserved and marked with *.Click here for file


Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish.krt4:NTR-hKikGR)cy17 (killer line), which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR+ fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR+ signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR+ fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR+ fluorescent signaling.This study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(The killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and provides a valuable tool to visualize and quantify the anti-apoptotic potential of interesting target genes in vivo. The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo. The epidermis is the outermost and largest structure covering the body. It plays a role in protection against mechanical injury forming a major barrier to deleterious environmental agents. The mammalian epidermis comprises four major layers: the basal layer, spinous layer, granular layer and cornified layer. The stem cells predominantly distribute in the basal layer and mitotic division of these cells generates other differentiated cells. In contrast to mammals, fish epidermis is relatively thin and simple. During early development, the fish epidermis has only two layers; the outermost enveloping layer (EVL) and inner epidermal basal layer (EBL), which provide a first-line defense system against pathogens. Fish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through the skin in vitro culture methods. However, some drawbacks such as dissimilarity to real skin physiology in vivo.When skin is exposed to environmental stress this triggers an apoptotic response to prevent accumulation of oncogenic mutations in its layers. Activation of repairing pathways ensues in order to recover skin integrity in vivo is possible by adapting a similar approach to that used in the mouse skin system.The zebrafish is a powerful model organism that has been extensively used to study developmental mechanisms, genetics, apoptosis, and human disease in recent years Escherichia coli converted into toxic form when exposed to Met. Metabolic Met behaves as a DNA interstrand cross-linking agent to exert a cytotoxic effect and induce apoptosis. Recently, the wild-type NTR protein has been engineered into a more powerful version, by codon-optimization krt4:NTR-hKikGR)cy17 (killer line) was established which overexpressed the NTR-hKikGR fusion protein under the control of the skin-specific krt4 promoter. Epidermal apoptosis could be induced by exposing the killer line embryos or adults to Met. Apoptotic cell death specifically occurred in the NTR-hKikGR-expressing cells, accompanied by the loss of fluorescent appearance. The zebrafish binary system, which combines killer and testing lines, therefore provides a unique quantitative and fast tool to study the signaling molecules which modulate skin apoptosis in living animals.This study aimed to a create a specific platform to conduct skin ablation by generating a transgenic zebrafish with a skin-specific promoter to drive nitroreductase (NTR), and then adding metrodinazole (Met) to cause specific ablation of superficial skin cells. NTR is a non-toxic reductase isolated from + skin cells widely distributed in the middle and basal layers, while the upper superficial layer displayed very few BrdU+ signals in most cases cy34 was initially created. It was confirmed that the zebrafish krt4 promoter could specifically drive nucleus-targeted EGFP cy34 demonstrated that the krt4 promoter can specifically target the superficial skin layer covering the scales cy17, killer line) displayed the strongest fluorescent signal and exhibited normal skin development, with no evidence of cell toxicity in the absence of prodrug treatment. It was, therefore, selected to generate the homozygotic line for further skin ablation studies. In the killer line embryos, detection of the NTR-hKikGR+ signals first occurred at the 5-somite stage (data not shown). NTR-hKikGR+ signals in the skin gradually up-regulated and finally displayed robust expression in the pharyngeal arch epithelium cy34, whose skin nucleus was highlighted with nlsEGFP fluorescent signals. Results showed that the NTR-hKikGR+ signals were located adjacent to the nuclei of EVL in the compound transgenics , the most sensitive version of NTR tested ithelium . The flunsgenics . This unpromoter .+ signals were monitored and photographed every 24 hours until 96 hpf. In the absence of Met (untreated), NTR-hKikGR+ fluorescent signals were robust throughout the entire experiment  activated Met suppressed the krt4 promoter and blocked the expression of NTR-hKikGR+ fusion protein; (2) activated Met caused redistribution of NTR-hKikGR+ fusion protein back to the cytoplasm compartment causing it to lose its aggregated, high fluorescent nature; (3) activated Met triggered cell apoptosis in skin. The lack of significant difference in nlsEGFP+ cells between untreated  and Met-treated Tg(krt4:nlsEGFP)cy34  ruled out the hypothesis that the same krt4 promoter also drives the nlsEGFP reporter gene . Compared to embryos, the adult killer line displayed higher efficacy of NTR/Met-ablation system. Administration of Met concentration higher than 2.5 mM was too stressful and, therefore, lethal. Compared to the untreated group  from 10 to 100 µM to scavenge the oxidative stress , to potent alkylating agents which can diffuse and kill non-NTR expressing neighboring cells by a bystander effect + skin cells after ablation were calculated and statistically compared at 48 hpf by one-way ANOVA. In the untreated killer line , mouse constitutively active Stat3 (Stat3), or HPV16 E6 (E6) in a superficial skin-specific manner. Previous studies documented the attenuation of UVB-induced apoptosis in skin culture cells or genetic modified mice by the three testing genes ler line  or untreler line , the denn = 134, . Upon exkrt4 promoter, were injected into homozygotic killer lines embryos and then skin ablation was triggered by following the protocol used in stable transgenics. Consistent with results obtained from the stable lines, the transient overexpression of myrAkt1  and reverse primer (GGGGACTGCTTTTTTGTACAAACTTGCCGGATCCTGTGTCTTTGAGTTGC-3′5′-). For p5E entry cloning, the attB4 and attB1r sites were added for forward and reverse primers, respectively, at the 5′end of primers and highlighted by underlines. The PCR products were then cloned into pDONRP4-P1R (Invitrogen) by BP reaction to obtain p5E-krt4. The resulting p5E-krt4 vector contained 2.2 kb upstream regulatory sequences of krt4 gene sufficient to target transgenes specifically expressed in skin cells GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGATATCATTTCTGTCGC-3′5′-) and reverse primer (5′-CGCTGGTGATCACGCTCACCATCACTTCGGTTAAGGTGATGTTT-3′). The hKikGR open reading frame was amplified from plasmid phKikGR1-MN1 5′-AAACATCACCTTAACCGAAGTGATGGTGAGCGTGATCACCAGCG-3′) and reverse primer (GGGGACCACTTTGTACAAGAAAGCTGGGTTTACTTGGCCAGCCTGGGCAGGC-3′5′-). The two primary PCR products were mixed and subjected to a secondary round of PCR with forward primer (GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGATATCATTTCTGTCGC-3′5′-) and reverse primer (GGGGACCACTTTGTACAAGAAAGCTGGGTTTACTTGGCCAGCCTGGGCAGGC-3′5′-). The attB1 and attB2 sites were added for forward and reverse primers, respectively, at the 5′end of primers and highlighted by underlines. The final PCR products were cloned into pDONR221 (Invitrogen) to generate pME-NTR-hKikGR. Finally, p5E-krt4, pME-NTR-hKikGR or pME-nlsEGFP Tol2 kit in vitro using pCS-transposase plasmid (kindly provided by Dr. Koichi Kawakami) as a template. DNA was linearized with NotI at 37°C overnight and cleaned up using DNA Clean/Extraction Kit . Capped mRNA was synthesized using mMESSAGE mMachine SP6 Kit (Ambion). For generation of transgenic zebrafish, expression constructs of pTolDest-krt4-NTR-hKikGR-pA (50 ng/µL) or pTolDest-krt4-nlsEGFP-pA (50 ng/µL) were mixed with in vitro transcribed transposases mRNA (50 ng/µL), and approximately 1–3 nL DNA solution was injected into the animal pole of one-cell stage embryos. The injected embryos were raised to adulthood and the putative founders were screened according to the green fluorescent signals in the skin of their F1 progenies. The detail procedures to generate Tg(krt4:Hsa.myrAkt1)cy18 (testing line to express human constitutively active Akt1), Tg(krt4:Mmu.Stat3)cy6 (testing line to express mouse constitutively active Stat3), Tg(krt4:Hpv.E6)cy38 (testing line to express HPV16 E6) will be available upon request. For assay of gene function at F0 generation, 5 nL of a solution containing expression constructs of either pTolDest-krt4-myrAkt1-pA, pTolDest-krt4-stat3-pA, or pTolDest-krt4-HPV16 E6-pA at 50 ng/µL concentration, together with transposases mRNA at 50 ng/µL concentration, was injected into killer line embryos at one-cell stage. The transgenic fish line nomenclatures were approved by the Zebrafish Nomenclature Committee of ZFIN (http://zfin.org). All experiments were approved by the animal use committee at Chung Yuan Christian University .Transposase RNA was synthesized Metronidazole  was dissolved in double distilled water as a 20 mM stock and stored at −20°C in the dark. For skin ablation/recovery at embryonic stage, at least twenty wild-type or transgenic zebrafish embryos aged at 24 hpf were incubated with 10 mM Met solution (diluted with fish water) at 28.5°C. At indicated times Met solution was removed and replaced with Met-free fish water for recovery. For adult skin ablation/recovery, wild-type and transgenic fish were incubated with 2.5 mM Met solution for 3 days at 27°C, then Met was withdrawn and replaced with Met-free fish water for recovery. At each time point, at least five fish were deeply anesthetized using 0.03% MS222 and sacrificed for histological assessment.For plastic section, embryo or adult zebrafish were fixed in 4% paraformaldehyde/PBS for 1 day, washed with PBST and then dehydrated with 100% methanol for 1 day. After completely dehydration, samples were infiltrated and embedded in Technovit 7100 resin (Heraeus Kulzer). Samples were sectioned at 1–2 µm intervals and stained with hematoxylin and eosin staining kit (Merck). For paraffin section, adult zebrafish were fixed in 4% paraformaldehyde/PBS for 1 day and transferred to Davidson's solution , 10% Acetic acid, 20% Formalin, and 30% double distill water) for 3 days at room temperature. This procedure decalcifies the hard tissues and also keeps the skin in good morphology. The decalcified samples were then dehydrated in ascending ethanol, cleared with Neo-clear (Merck) and embedded in Paraplast Plus/Paraplast HM (Leica) in a 7∶3 ratio (vol/vol). The paraffin embedded tissues were sectioned at 5 µm intervals with a rotational microtome  and then stained with H&E or PAS staining kit according to the manufacturer's instructions (Merck). For immunohistochemistry, the deparaffined slides were treated for antigen retrieval by incubating with 1 mM EDTA (pH 8.0) solution Adult transgenic zebrafish were systematically incubated in 10 mM BrdU /5% DMSO solution for 24 hr and then transferred to fresh fish water for 1 hr to remove the unincorporated BrdU. After performing fixation, decalcification and paraffin section processes according to the protocol mentioned previously, the de-paraffined slides were depurinated with 2.5N HCl for 30 min at room temperature and immersed in 0.1 M borate buffer for 10 min to enhance the detection sensitivity for the BrdU-positive cells in skin. The depurinated slides were then subjected to antibody staining with mouse anti-BrdU  and HRP-conjugated goat anti-mouse IgG (Jackson ImmunoResearch).+-K+-ATPase , rabbit anti-Atlantic salmon anterior gradient 2 Zebrafish embryos aged at 48 hpf were fixed in 4% paraformaldehyde/PBS for 12 h at 4°C. After extensive washing in PBST, embryos were transferred to 100% methanol at −20°C for 2 h and subsequently subjected to rehydration with PBST. After blocking with 3% BSA/PBST at room temperature for 60 min, embryos were incubated at 4°C overnight with 1∶200 diluted primary antibodies as follows: mouse anti-human p63 , mouse anti-chicken NaTail fins dissected from 15 individuals of wild-type or killer lines were pooled and homogenized in protein extraction solution . The total lysates were centrifuged at 13,000 rpm for 20 min at 4°C to remove cell debris, and the supernatant was collected for further study. The protein concentration was determined using BCA protein assay kit  and detected using Synergy HT Multi-Mode Microplate Reader . Total cellular proteins of 35 µg were separated by 12.5% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Following incubation with blocking solution, PVDF membranes were incubated with primary antibodies overnight and then HRP-conjugated secondary antibodies (1∶1000 dilution) for 1 hr at room temperature. The primary antibodies and their dilution factor were as follows: mouse anti-hKikGR , rabbit anti-activated caspase 3 , mouse anti-pan cytokeration , rabbit anti-human phosphorylated GSK3α/β (pY279/pY216) , rabbit anti-human phosphorylated stat3 (pY705) , rabbit anti-human phosphorylated p70 S6 kinase (pT421/pS424) , and mouse anti-β actin . After a series of washing, locations of protein were revealed by incubating in the WEST-ZOL PLUS solution  for 2 min at room temperature in the dark room, and the images were acquired using FUJIFILM LAS 3000 Imaging Analyzer .2O2/MeOH for 10 min at room temperature. Subsequently, embryos were rinsed two times with PBS and incubated with labeling solution, 10 µL of enzyme solution plus 90 µL of label solution at 37°C for 2 h following the kit instructions (Roche Applied Sciences). Embryos were then washed three times in PBS, for 5 min each time, at room temperature. Subsequently, embryos were incubated with 100 µL of converter POD at 37°C for 30 min. Embryos were rinsed three times in PBS and incubated with DAB solution  for 5 min.For TUNEL assay, embryos aged at 18 hpf were pre-treated with PTU and then processed to PFA fixation at 48 hpf. Following washing in PBST for 10 min they were then stored in 100% methanol at −20°C for over 2 h. Embryos were incubated with 3% HThirty embryos aged at 3 dpf from wild-type or testing lines were collected and homogenized in RNAzol RT  with Bullet Blender  tissue lyser to isolate total RNA according to the manufacturer's instructions. Total RNA concentration was determined by spectrophotometry, and the RNA quality was checked by running electrophoresis in RNA-denatured gels. For RT-PCR, 1 µg of total RNA was reverse-transcribed with RevertAid first cDNA synthesis kit  and then PCR was performed with SYBR green dye according to the manufacturer's instructions. The primer sequences used to perform RT-PCR and the PCR amplicon size are listed in tp53 MO , at a concentration of 5 ng/embryo, was injected into yolks at the one-cell stage. The specificity and efficacy of tp53 MO has been validated previously To achieve the maximal knock-down effect, + cells, the original images were processed using Photoshop CS3 software to select a region of interest (ROI) at 300 µm×150 µm dimensions. The total cell number in this ROI was calculated using ImageJ software (http://rsbweb.nih.gov/ij/) and statistically compared using t-test or one-way ANOVA.Representative DAB-stained or fluorescent images were acquired using an upright microscope  equipped with a digital camera  or a dissecting microscope  equipped with a cool CCD (Evolution VF). For quantifying the relative density of skin cells or TUNELFigure S1Validation of the transmission, expression and function of testing lines in stable transgenics using genotyping, RT-PCR and Western blot analysis. Assay of the stable transmission and expression of transgenes in different testing lines at the DNA and mRNA levels by genotyping (A) and RT-PCR (B), respectively. The relative position of the primers used to perform genotyping or RT-PCR is illustrated by red arrows. The predicted amplicon size is also illustrated in the right panel. The plasmid architectures were highlighted in the upper panel. (C) Western blot assay on protein lysates extracted from adult tail fins showing highly phosphorylated Akt downstream targets of GSK3α/β and 70S6K in myrAkt1 testing line. β-actin served as a protein loading control. (D) Quantitative real-time RT-PCR showed that overexpression of pro-survival genes of Stat3 down-regulates the pro-apoptotic genes bida and puma in transgenic embryos aged at 48 hpf. (E) Quantitative real-time RT-PCR showed that overexpression of virus oncogene of HPV16 E6 up-regulates the cell cycle-related genes ccne and cdk2 in transgenic embryos aged at 48 hpf.(PDF)Click here for additional data file.Table S1The PCR amplicon size and primer sequences used to perform RT-PCR.(XLSX)Click here for additional data file.Movie S1Swimming behavior assessment of killer line treating without  or with . Met, metrodinazole.(WMV)Click here for additional data file.


Drosophila, and ortholog to M6a. Unexpectedly, we found that decreased expression of M6 leads to female sterility. M6 is expressed in the membrane of the follicular epithelium in ovarioles throughout oogenesis. Phenotypes triggered by M6 downregulation in hypomorphic mutants included egg collapse and egg permeability, thus suggesting M6 involvement in eggshell biosynthesis. In addition, RNAi-mediated M6 knockdown targeted specifically to follicle cells induced an arrest of egg chamber development, revealing that M6 is essential in oogenesis. Interestingly, M6-associated phenotypes evidenced abnormal changes of the follicle cell shape and disrupted follicular epithelium in mid- and late-stage egg chambers. Therefore, we propose that M6 plays a role in follicular epithelium maintenance involving membrane cell remodeling during oogenesis in Drosophila.We had previously shown that the transmembrane glycoprotein M6a, a member of the proteolipid protein (PLP) family, regulates neurite/filopodium outgrowth, hence, M6a might be involved in neuronal remodeling and differentiation. In this work we focused on M6, the only PLP family member present in  M6a mRNA levels in the hippocampus, and this downregulation is prevented by chronic administration of antidepressants www.genecards.org).The glycoprotein M6a was identified years ago, and despite its prominent expression in the central nervous system and other tissues M6a belongs to the myelin proteolipid protein (PLP) family. In mammals, other members of this family include the closely related M6b and the founder PLP, with its splice variant DM20. All of the PLP members have four transmembrane domains that allow their localization at the plasma membrane and are widely conserved along evolution from arthropods to mammals plp genes , a single M6 gene is found in arthropods M6 gene resembles that of the mouse M6a gene, but also the predicted M6 protein shares amino acid similarity with the mouse M6a Drosophila as the model system.While mammals contain three D. melanogaster, induced by M6 downregulation using hypomorphic alleles and RNAi lines. Such sterility was associated with egg permeability and collapse, suggesting M6 involvement in eggshell biosynthesis. We found that M6 is expressed in the follicular epithelium of egg chambers throughout oogenesis in wild type flies. Tissue-specific M6 knockdown targeted to follicle cells impaired follicle cell shape changes and triggered disruption of the follicular monolayer. Indeed, partial loss of M6 function unraveled its key role at stages where cell adhesion, cytoskeletal rearrangements and cell-cell communication enable cell shape changes and organization.In this work we focused on a phenotype, namely female sterility in Flies were grown and maintained at 25°C in vials containing standard cornmeal-agar medium.1118w stock was used as the wild-type control. Potential M6 (CG 7540) mutant stocks 1 w67c23y; P{EPgy2}M6EY07032, 1118w; P{GT1}M6BG00390 and 1118w; Mi{ET1}M6MB02608/TM3, Sb1 Ser1 were renamed M601, M602 and M603, respectively, and were obtained from the Bloomington Stock Center, along with the GAL80ts lines. The CA06602 stock (M6GFP) was obtained from the GFP Protein Trap Database at the Carnegie Institution M601, M602 and M6GFP strains were backcrossed several generations to 1118w to minimize background effects. The follicular GAL4 driver PG45.G4/FM7 was provided by Dr. Stein. Fly strains, such as 198Y-GAL4 (P{GawB}198Y), e22c-GAL4 (P{en2.4-GAL4}e22c), 55B-GAL4 (P{GawB}55B), da.G32-GAL4 (P{GAL4-da.G32}UH1), slbo.2.6-GAL4 (P{GAL4-slbo.2.6}1206), c355-GAL4 (P{GawB}c355), c204-GAL4 (P{GawB}c204), T155-GAL4 (P{GawB}T155), c329b-GAL4 (P{GawB}c329b), tubP-GAL4 (P{tubP-GAL4}LL7), and UAS-CD8GFP were obtained from the Bloomington Stock Center. The UAS-M6-RNAi strains employed were: ID 7540R-1 and 7540R-2 from the National Institute of Genetics Fly Stock Center (NIG-Fly) termed here as M6-RNAiNIG (only the first one was shown), and 101757 from the KK-library Vienna Drosophila RNAi Center (VDRC) termed here as M6-RNAiVDRC. Experiments with the TARGET system A M6 mutant alleles were generated by excision. The original P-element (EY07032) from M601 was removed with the transposase (Δ2–3). White-eyed stocks corresponding to independent excision events were kept as M6Δ01 allele strains. To characterize the M6 allelic series, PCR reactions on genomic DNA from homozygous flies were performed as detailed in M6 alleles, or at 18/29°C in M6 knockdown experiments. The number of adult offspring per vial resulting from each cross was quantified.Female fertility was assessed crossing two females  to 5 wild type males per vial. For male fertility experiments, 5 males were crossed to 2 wild type females. Crosses were kept in each vial with yeast paste for 2–3 days at 25°C when testing the fertility of Egg laying was analyzed in batches of 30 or 50–60 females with wild type males when incubated at 25°C or 18/29°C respectively. Females were allowed to lay eggs on yeasted agar plates for 6 hours at 25°C or for 4 hours at 18/29°C. Only eggs laid during the afternoon were scored in order to reduce variability due to rhythmic behavior Egg collapse was determined collecting 0–4 hours-old eggs and transferring them onto dry plates. Collapsed and non-collapsed eggs were counted every 5 minutes.Eggshell permeability was assessed by the neutral red assay  to isolate total RNA. Then PolyA+mRNA was purified using the PolyATract mRNA Isolation System . Complementary DNA was synthesized using oligo dT and SuperScript™ II Reverse Transcriptase enzyme (Life Technologies).Rp49 and gapdh as housekeeping genes. Values shown in all figures were calculated using Rp49 as the reference gene. Normalizations to gapdh resulted in almost identical patterns. Relative quantification was performed using a comparative CT method qPCRs were carried out in a 7500 Real-Time PCR System . Quantitation of each cDNA was achieved using SYBR Green PCR Master Mix (Applied Biosystems) in triplicate. Primer sequences are detailed in t test or one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison tests. Non-parametric analysis was performed  when assumptions on the normal distribution and variance did not allow otherwise.Graphs were generated with GraphPad Prism version 5.0 . Statistical analysis was performed with IS (Infostat software). Group means were analyzed for overall statistical significance by unpaired Student's M6 locus were identified (at http://flybase.org/); three potential M6 mutants, renamed M601, M602 and M603  and ubiquitous expression of M6-RNAi . To confirm that female sterility was associated to the insertion within the M6 locus we mobilized the P-element from M601, giving rise to M6Δ01-rev. Excision of M601 rescued fertility to wild type levels . Interestingly, ovaries from M602, the non-sterile strain, displayed elevated M6 levels.We anticipated that M6 should be present in the central nervous system based on information available on M6a  RT-qPCR  and founA levels , revealiM6, CG33214, is only 384 bp away from the insertion site of the P-element in M601. However, CG33214 mRNA levels in ovaries showed no differences between M601, M6Δ01-rev and control flies by RT-qPCR , indicating that subtle changes in M6 levels (right panel) associated with a severe decrease in female fertility. Male fertility did not show significant differences among M6 alleles (data not shown). Taken together, these results show that female sterility is triggered by M6 downregulation.Incomplete excision of the P-element in M601 females. The number of eggs laid by M601 flies was significantly reduced compared to controls  throughout oogenesis, implying it plays a role specific to the FE.M6 expression with the GAL4/UAS system M6-RNAi lines. Employing ubiquitous promoters, lethality became apparent during early development, when M6 localizes to the central nervous system and likely to the epithelia  maintained at 29°C and PG45>M6-RNAi at 18°C, a non-induced condition  did not exhibit female sterility defects at either temperature (data not shown). Thus, M6 knockdown in the FE triggered female sterility in a tissue specific manner.Since M6 is essential throughout development we resorted to an inducible system ondition . We thenc series , 2. PG45M6-RNAiNIG and control groups at 18°C. However, a clear increase in the percentage of permeable eggs became evident in those expressing the M6-RNAiNIG , suggesting a failure in egg chamber elongation.A closer inspection of stage 14 egg chambers from the PG45>M6 downregulation driven by other follicular GAL4 lines were unable to trigger female sterility. A detailed description of the GAL4 drivers and enhancer traps used and the phenotypes displayed are summarized in In addition, we found that M6-RNAiNIG-induced females. Loss of M6 triggered a number of developmental defects such as disorganization and even total disruption of the FE as revealed by the presence of gaps  led to similar phenotypes, indicating that egg permeability, altered follicular epithelium integrity and arrested egg chamber development were indeed the result of downregulated M6 expression in the FC. However, despite M6 downregulation in both M601 and M6-RNAi treated ovaries reached similar M6 mRNA levels (40–45%), the ovarian phenotypes displayed by M6-RNAi lines were stronger than M601, implying a nonlinear relationship between the two. Moreover, M6 locus gives rise to different M6 versions as a result of the use of alternative promoters and alternative mRNA splicing , underlying the complexity of this locus. We can envision at least two explanations to reconcile this apparent discrepancy. On the one hand, although both M6-RNAi putatively target all isoforms, their expression was limited to the PG45 domain. This might not completely describe the M6 endogenous pattern evidenced by the enhancer trap GFP-M6, which in turn reflects the pattern of isoforms driven by P1 (and not P2). Thus, it is possible that additional tissues (such as the germline) could contribute the remaining M6 levels detected by qPCR in whole ovarian extracts. On the other hand, the insertion in M601 could spare specific isoforms (such as those driven by P2), which could be differentially expressed in ovaries and contribute to the detected M6 levels. This topic is the focus of intense analysis and will be reported elsewhere .Using different genetic strategies such as the insertional mutants and M6 knockdown in the FE we demonstrated that M6 plays an essential role during egg chamber development. Interestingly, in addition to the egg permeability phenotype displayed by er 1 P1, , and thuM6-RNAi lines were first observed at stage 9. Thus, while oogenesis apparently proceeded relatively normally from germarium through stage 8, morphological defects triggered by downregulated M6 expression became detectable at later stages.Several evidences point to a temporal window for M6 requirement, which coincides with the time the FE undergoes tissue remodeling During the second half of oogenesis the FC have two main roles: the communication with the oocyte that is essential for patterning the egg chamber and early embryo, as well as the formation of the eggshell. The latter is characterized by a sequence of migrations and cell shape changes involving subpopulations of FC that cover the oocyte and synthesize specialized eggshell structures. During mid-oogenesis the epithelial FC cease proliferation (stage 7), and form a cuboidal epithelium surrounding the whole egg chamber (stage 8) M6 downregulation in FC triggered a dumpless-like phenotype. Although it usually implies structural defects in ring canals or actin-organization, it could also arise as a result of altered centripetal migration as observed at st10 late  is probably stored in microvilli. However, there is no membrane reservoir in the basal one; consequently, a portion of the lateral membrane previously engaged in cell-cell interactions called zone of contact (ZOC) is converted into basal membrane (open ZOC) and faces the basal extracellular matrix. Such plasma membrane remodeling strengthens adhesion and ensures epithelial integrity at the next stages of development t10 late  and suggDuring late oogenesis, the structural components of the eggshell are secreted by the FC surrounding the oocyte in a precisely regulated temporal and spatial manner. In normal eggs the vitelline membrane and the wax layer prevent dehydration. Abnormalities in the deposition of either one of these structures or, alternatively, a failure to completely cover the anterior end of the oocyte upon migration of the anterior FC might cause eggs to loose water and show a collapsed phenotype Drosophila gut and wing epithelia, integrins are implicated in the maintenance of columnar morphology In M6 downregulation is subtle, additional studies are needed to investigate whether M6 plays a role during the planar-polarized orientation of basal F-actin bundles in the follicular epithelium.Mutations that cause spherical egg phenotype are known to disrupt proteins mediating interactions between the actin cytoskeleton and the extracellular matrix M6 downregulation may be due to defects in cell adhesion leading to alterations in cell shape, which in turn disrupt the epithelial organization. Preliminary studies supporting this hypothesis suggest altered distribution of DE-cadherin at the tricellular junctions of M601 late stages egg chambers . A potential role of M6 in cell adhesion is further supported by the fact that PLP was proposed to act as an adhesion molecule Drosophila oogenesis, which is particularly relevant in light of the conservation of this protein between flies and mammals.The failure in the maintenance of epithelial architecture induced by Figure S1The P element insertion in M601 does not affect expression in the nearby CG33214 locus. The upstream locus contiguous to M6, CG33214, which transcription initiation is only 384 pb away from the insertion site in M601, was not affected by the P element insertion. Expression of CG33214 was measured in ovaries from wild type, M601 and M6Δ01-rev (imperfect excision) by RT-qPCR and normalized to Rp49. Mean ± s.e.m, n = 3. Statistical analysis included One Way Anova (p>0.05).(TIF)Click here for additional data file.Figure S2M601 ovarioles show neither gross morphological defects nor altered localization of vitelline membrane biosynthesis markers. Immunofluorescence of wild type  and M601 (lower panel) egg chambers with antibodies directed to Armadillo (A), Fasciclin III , sv23 , Cad99C  and Yolkless . (B–E) Egg chambers costained with phallloidin to visualize F-actin (red). Scale bar is 50 µm, unless otherwise indicated.(TIF)Click here for additional data file.Figure S3M601 and M601/M603 egg chambers do not show altered localization of vitelline membrane proteins. (A) Eggs laid by heterozygote M601/+ and M603/+ and transheterozygote M601/M603 females were collected and incubated with the neutral red dye. All (100%) M601/M603 mutant eggs were permeable, which is taken as an indication of an abnormal vitelline membrane. (B–D) Immunofluorescence of wild type , M601 (middle panel) and M601/M603 (right panel) sectioned egg chambers with antibodies directed to sV23 , sV17  and Yolkless . Numbers indicate egg chambers stages. Nuclei are visualized in red (DAPI). Projection of z-stacks are shown in (Cc.) and . M6 mutant egg chambers displayed a continuous line of the vitelline membrane proteins (sV17 and sV23 staining), and no relocalization into the oocyte. Scale bar is 50 µm. Oo, oocyte; Fc, follicle.(TIF)Click here for additional data file.Figure S4GFP-M6 colocalizes with Armadillo in the follicular epithelium. (A) Female fertility assessment of M6GFP flies. Female fertility was measured as the number of offspring per vial obtained when crossed to wild type males. Mean ± s.e.m, n = 3; unpaired t-test with Welch correction, p<0.05. (B) M6 mRNA levels in ovaries was measured in wild type (w) and M6GFP flies by RT-qPCR and normalized to Rp49 or gapdh. Statistical analysis included an unpaired t-test with Welch correction, p>0.05; mean ± s.e.m, n = 3. (C) Armadillo/β-catenin labels the membrane of follicle and nurse (to a lower extent) cells (magenta). GFP-M6 is localized to the membrane of the follicle cells throughout oogenesis in M6GFP (green). Colocalization is shown on the right panel. Representative egg chambers are shown. F-actin was labeled with phalloin (red) and nuclei with DAPI . Scale bar is 20 µm, unless otherwise indicated.(TIF)Click here for additional data file.Figure S5GFP-M6 colocalizes with Fasciclin and Cad99C. GFP-M6 (green) localizes to polar  and border cells (A) and to microvilli of the follicle cells labeled with anti-Cad99C antibodies  in M6GFP. Colocalization is shown on the right panel. Representative egg chambers are shown. F-actin was labeled with phalloidin (red) and nuclei with DAPI . Scale bar is 20 µm.(TIF)Click here for additional data file.Figure S6M6 requirement during development. (A) GFP-M6 is expressed in the embryo. Immunofluorescence images at different depths from the same M6GFP embryo are presented in the upper and lower panels. Fasciclin II  labels longitudinal axon fascicles in the nervous system. GFP-M6 (green) is expressed in the longitudinal fascicles as shown by the colocalization (right panel), as well as in other tissues such as the epithelium (lower panel). Therefore, M6 knockdown in the embryo might be responsible for early lethality , an arrested pupae development was observed, suggesting that in addition to driving expression in follicle cells, those GAL4 drivers are also expressed at an unspecified tissue during larval/pupae formation. Thus, M6 deprivation during larval or pupae development stages abrogates metamorphosis  and control flies (M6-RNAiNIG with no driver). Pupae arrest was detected during metamorphosis while control flies developed normally. Similar results were observed using T155. Scale bar is 2 mm.see also . Images (TIF)Click here for additional data file.Figure S7Follicular GAL4 drivers used to downregulate M6 in FC. Representative images of immunofluorescence of (A) 55B>CD8GFP, (B) da.G32>CD8GFP, (C) slbo>CD8GFP, (D) PG45>CD8GFP, (E) c355>CD8GFP, (F) c204>CD8GFP, (G) T155>CD8GFP, (H) c329b>CD8GFP and (I) tubP>CD8GFP ovarioles. GAL4 expression is revealed by CD8-GFP labels (green) in follicle cells. Nuclei were stained with propidium iodide or DAPI (red). PG45 expressed GAL4 in all follicle cells throughout oogenesis (D). Note GAL4 expression in subpopulation of follicle cells with different drivers . Interestingly, those follicular drivers  did not trigger any female sterility in the context of M6-RNAi, whereas PG45 did Click here for additional data file.Figure S8M6 downregulation in follicle cells induces a variety of defects in mid- and late oogenesis. Confocal images of representative egg chambers at different stages of oogenesis. Ovaries were dissected and stained with phalloidin (red), DAPI (blue) and Armadillo (green). PG45>M6-RNAiNIG  flies were raised at 18°C and then adult females were transferred to 29°C for 24, 48 or 72 hours to induce M6 interference in the follicular epithelium or kept at 18°C as the non-induced control. Control females, M6-RNAiNIG (without PG45), shifted to 29°C for 72 hours were undistinguishable from non-induced controls (data not shown). Egg chambers corresponding to stages 4–7 and 9 (A), late stage 9, stage 10A and 10B (B), stages 10 and 11 (C), stages 12 and 13 (D) and stage 12 (E) are shown. (A) No morphological defects were observed at early stages of oogenesis. The presence of gaps in the follicular epithelium after 48 h of M6-RNAi induction became detectable from stage 8 onwards (indicated by white arrowheads), and from stage 7 onwards after 72 h of induction. Note defects in the FC shape. (B) The first defects observed after a 24 hour induction involved follicle cell morphology in egg chambers from mid-oogenesis, including a deficit in the thickness gradient of the posterior follicle cells in late stage 9 along with a failure in the columnar shape of the follicle cells in egg chambers reaching stage 10A. Defects in the overall shape of the follicle cells are indicated by yellow asterisks. Magnified views of the indicated areas are shown at the bottom of each egg chamber. White arrows highlight morphological hallmarks of the dumpless-like phenotype in st10B . (C) After a 24 hour induction, defects in centripetal migration were detected in stage 10–11 egg chambers as indicated by yellow arrowheads. This alteration might cause the dumpless-like phenotype(white arrow). (D) Disorganization of the follicular epithelium induced in stage 11–13 egg chambers after 24 hours associated with abnormal Armadillo accumulation at the tricellular junctions. (E) At later stages of oogenesis, such as stage 12, gaps were evidenced after 48 h of induction (indicated by white arrowheads). The dumpless-like phenotype (white arrow) is detected likely as a result of altered centripetal FC migration (yellow arrowhead). Scale bar is 50 µm.(TIF)Click here for additional data file.Table S1Primer sequences used in PCR. To characterize the M6 allelic series, PCR reactions on genomic DNA obtained from homozygous flies were carried out with primers directed to the 5′UTR of M6A flanking the original P element insertion. The E-GFP fusion to M6 was assessed by a Hemi-Nested PCR experiment employing cDNA from M6GFP ovaries. Two rounds of PCR were performed in order to amplify isoforms with low representation. A second PCR experiment was carried out employing different pairs of primers. M6 mRNA isoforms tagged to the C-terminus of E-GFP were determined by sequence alignments . The sequences of the expressed GFP::M6 proteins were predicted in silico. Primer sequences used in qPCR were designed using Primer Express 3.0 software (Applied Biosystems) and targeted to the 3′UTR of all M6 mRNA isoforms, glyceraldehyde phosphate dehydrogenase (gapdh) and ribosomal protein 49 (Rp49). To confirm M6 levels in the M6 allelic series a second pair of primers for M6 3′UTR was used.(DOC)Click here for additional data file.Table S2M6 knockdown triggers lethality during development and sterility in adult females. The table summarizes both the developmental arrest phenotype (middle column) and the female sterility phenotype (middle column) induced by M6-RNAi. Different GAL4 drivers and enhancer trap lines (left column) were used to express two independent UAS-M6-RNAi lines from the NIG-fly  and VDRC  stocks centers. Interference was amplified including UAS-Dicer2 (from VDRC). Early development arrest is triggered upon M6 downregulation. When pleitropic GAL4 drivers such as tubP-GAL4 M6-RNAi) lethality became apparent at early stages during development, suggesting an essential role for M6 early on. This was also evidenced by the lethality of the M603 mutant and a number of M6 alleles generated by P element excision of the M601 mutant (data not shown). Some of the drivers required the addition of an allele of UAS-Dicer2 to trigger the same interference with both RNAi stocks (data not shown). Only complete female sterility was scored as female sterility. Although differences in the strength of the GAL4 drivers cannot be ruled out, M6 downregulation induced female sterility only when M6-RNAi was expressed in all follicle cells from st7 onwards . Follicular GAL4 drivers were crossed to UAS-CD8GFP to check GAL4 expression in follicle cells  tested with M6-RNAiNIG. (***) tested with M6-RNAiVDRC FC for follicle cells.(DOC)Click here for additional data file.


This higher replication capacity does not lead to a higher cytotoxicity compared to previously described subtype C viruses. In addition, the pZAC Vpu is also shown to be able to down-regulate CD4, but fails to fully counteract CD317. The Human Immunodeficiency Virus type 1 (HIV-1) subtype C is currently the predominant subtype worldwide. Cell culture studies of Sub-Saharan African subtype C proviral plasmids are hampered by the low replication capacity of the resulting viruses, although viral loads in subtype C infected patients are as high as those from patients with subtype B. Here, we describe the sequencing and construction of a new HIV-1 subtype C proviral clone (pZAC), replicating more than one order of magnitude better than the previous subtype C plasmids. We identify the  HIV-1 subtype C has unique characteristics: These include the presence of an additional NF-κB enhancer binding site in the long terminal repeat (LTR), a prematurely truncated Rev, and an insertion of five amino acids at the amino terminus of Vpu [The genetic subtype distribution of HIV-1 group M, currently responsible for the majority of the AIDS pandemic, has become dynamic. The HIV-1 group M has been divided into nine subtypes, 51 circulating recombinant forms and numerous unique recombinant forms. From 2004–2007, subtype C accounted for nearly half (48%) of all global infections [s of Vpu ,4. + T cells and peripheral blood mononuclear cells (PBMCs) compared to subtype B clones, such as pNL4-3 [There are currently five HIV-1 subtype C proviral clones described, two from Sub-Saharan Africa ,6, two fs pNL4-3 ,13. env region of pMJ4 with that of our primary isolate . The phylogenetic relationships of the full-length infectious HIV-1 subtype C clones were analysed by constructing a neighbour-joining tree  of 0.05) were cultivated on PBMCs for up to 8 days . Viral t490 = 1.48 ± 0.049 pMJ4, OD490 = 1.32 ± 0.05; uninfected cells, OD490 = 1.36 ± 0.1). This experiment demonstrated that the infectious subtype C viruses did not affect the cellular metabolism in a significant way.It has been reported that the HIV subtype C displays a lower cytopathic effect than other subtypes. Since our cloned virus replicates at a higher titer than the previously characterised pMJ4, we analysed whether the low cytopathic effect was conserved with pZAC. To determine cell damage and elimination cause by pMJ4 and pZAC viruses, TZM-bl cells were infected with a MOI of 0.25 in triplicate assays. The cell survival and cellular metabolism was measured after two days by a cell proliferation assay according to the manufacturers instructions. The MTT values of both viruses were found to be in a similar range  were pre-incubated with 10 ng/mL of the CCR5 inhibitor Maraviroc for one hour. Cell culture supernatants were titrated on these target cells (MOI: 2) (Since a determinant for the higher pZAC titers is located in the n detail . We dete(MOI: 2) . The T-tenv fragment exchanged in the pMJ4/ZAC clone, we analysed Vpu function to antagonize CD317. CD317 was shown to inhibit virus release [vpu-ATG deficient pNL4-3∆vpu served as control. Viral titers were determined by titration on TZM-bl cells 2 days post transfection. pZAC derived titers were significantly reduced by CD317 indicating that pZAC Vpu is unable to significantly counteract CD317 , while pMJ4 titers remained unaffected   or pZAC vpu (3 µg) (p = 0.09), whereas pZAC Vpu failed to counteract CD317 completely . These results show that the ability of pZAC Vpu to counteract CD317 is impaired compared to pMJ4 Vpu.By analysing the pZAC sequence we discovered a divergent evolution of the Vpu amino acid sequences compared to the complete viruses A. The Af release . The pro = 0.07) B. In ordL4-3∆vpu  and pMJ4u (3 µg) C. Viral Vpu was reported to down-regulate CD4 as well as counteract the effects of CD317 ,22,23. Ivpu start codon. Functional analysis of one of these patient derived sequences showed that no functional Vpu was expressed (data not shown). So far only HIV-1 subtype N and P have been shown not to counteract CD317 by Vpu [env region were able to grow. The same holds true for the pcMJ4/ZACenv, which expresses only the env-region of pZAC in a pMJ4 background. These data raise the question, whether pZAC titers would further increase if the vpu sequence were replaced by sequences from ZAC.The surprising result of this study was that the pZAC Vpu fails to fully counteract CD317, which might be an immanent problem of other subtype C viruses as well. By analysing patient derived sequences from South Africa, we have observed that about 1% of these viruses showed a deletion in the 7 by Vpu ,26. Unfo7 by Vpu . The virPatient data: The retrovirus cohort represents patient samples diagnosed with either HIV or HTLV infection of which samples (plasma and serum) were stored from 1984 to 1995 at the Tygerberg hospital in Cape Town, South Africa. The patient, a South African coloured (mixed race) male born on 22 August 1931 was diagnosed with lymphocyte depleted Hodgkin’s lymphoma on 02 March 1989 and diagnosed as HIV-1 positive on 09 March 1989. He travelled frequently to Lusaka, Zambia, where he possibly became infected with the virus. env gene was amplified by PCR, sequenced and identified as subtype C [Subsequently, serum and peripheral blood mononuclear cells (PBMCs) were obtained during November 1989 (harvested on 20 and 21 November 1989) and the virus was co-cultured with PBMCs and isolated. High molecular weight DNA was extracted from the HIV positive cultures through phenol-chloroform extraction and stored. HIV-1 positive cultures were confirmed by reverse transcriptase (RT) assay that ranged from 12,495 to 35,073 counts per minute per milliliter (cpm/mL). The ubtype C .Exchange of the 5'-U3 promoter with CMV-IE-promoter: All PCR amplifications during this study were done with the stable proofreading Herculase II polymerase (Stratagene). Briefly, the HIV-1 subtype C promoter was replaced and cloned into the pMJ4 vector by overlapping PCR, using restriction sites (GCCGGCTAGTTATTAATAGTAATCAATTACGGGTC-3'), containing a NgoMIV restriction site, underlined in the sequence and CMV_overlap_R (5'-GTGCTCCCGGGCTCAGATCTGGTCTACCTAGAGAGACCCGGTTACTAAACCAGCTCTG-3'), containing the last 30 bp of the CMV promoter and the first 30 bp from HIV-1 subtype C transcription start (R-region). The 1.0 kb HIV-1 subtype C region from R-start to the SpeI site in gag was amplified from pMJ4 with primers CMF_overlap_F (5'-CAGAGCTGGTTTAGTAACCGGGTCTCTCTAGGTAGACCAGATCTGAGCCCGGGAGCTC-3') and HIVC_SpeI_R (5'-CTATTTGTTCCTGAAGGGTACTAGTGTTCCTGCTATG-3'), the SpeI site is underlined. Primers CMVstart_NgoMIV and HIVC_SpeI_R were then used to PCR amplify the 1.6 kb fragment and cloned directly into pMJ4. The presence of the CMV promoter was confirmed by DNA sequencing. The resulting plasmid was abbreviated as pcMJ4.on sites . The 600Cloning of pZAC: The cloning strategy used during this study is described in env of pMJ4 with that of our primary isolate, ZAC using standard cloning techniques. The 3.2 kb PCR product was amplified from the HMW DNA of the patient with primers containing the restriction enzyme recognition sites for PacI and BspEI (for primer sequences see gagpol and LTR-gag region. The gagpol region was replaced using restriction sites SpeI (corresponding to position 1507 of HXB2) and PacI (corresponding to position 6198 of HXB2), while the CMV-IE LTR-gag sequence was added as for pcMJ4. The new proviral clone was designated pcZAC. The 3'‑U5 was replaced using BspEI and the vector located NotI restriction site and the 5'-U3 CMV-IE was replaced with the pZAC derived 5'-U3 sequence. The final clone (without the CMV-IE promoter) was named pZAC. The pZAC sequence was submitted to GenBank: Acc. No. JN188292.Sequence and phylogenetic analyses: Full-length HIV-1 genome sequencing was done with a set of primers previously described [escribed . Sequencescribed . Phylogeescribed .Isolation of PBMCs and maintenance for cell cultures: PBMCs were extracted from donor EDTA blood by density gradient centrifugation with Histopaque®-1077 (Sigma-Aldrich). PBMCs were cultivated overnight at 37 °C at 5% CO2 with RPMI-1640 media stimulated with phytohemagglutinin-P, (PHA-P) (Sigma-Aldrich) (0.5 mg/mL) and human Interleukin-2 (IL-2) (Sigma‑Aldrich) (0.1 mg/mL) before being used for viral kinetics assays. HEK 293T and TZM-bl cells were maintained in minimal essential media (MEM) (Invitrogen) at 37 °C at 5% CO2. Cellular metabolism and cell viabilty was detemined with the cell proliferation assay (Promega) 2 d post infection. In brief: 20 µL of the indicator solution was added to 100 µL cultures. The cells were further incubated in the cell culture incubator for 90 min. The reaction was stopped by the addition of 25 µL 10% SDS and the absorbance was measured at 490nm. Uninfected cells served as control. Transfection of cell lines: For the infectious clones approximately 4 μg of plasmid DNA was transfected with the Fugene®6 transfection reagent (Roche Diagnostics) and the culture incubated for 2 to 3 days to allow for the expression of proteins.Infectivity assays: To analyse the infectivity cell culture supernatants were titrated on TZM-bl cells in triplicate assays as described before [lacZ gene. Two days post infection TZM-bl cells were fixed with ice-cold methanol/acetone for 5 min followed by a β-galactosidase stain, using X-gal as substrate. Blue cells were counted and the viral titers were calculated. The number of GFP expressing cells was determined after two days using a fluorescence microscope. Viral titers were subsequently calculated. A paired two-sample t-test was used to obtain the p-values given in the manuscript.d before . In brieCD4 determination: HEK 293T cells were cotransfected with the 2 µg of pMSCCD4 expression, 0.5 µg peGFP and 2 µg of the pZACvpu or pMJ4vpu expression plasmids. Cells were harvested and washed with FACS buffer. The monoclonal anti-human CD4 antibody (BioLegend) was diluted 1:25 and cells were stained for 15 min at 4 °C. The cells were subsequently fixed with Fix & Perm reagent (Invitrogen). CD4 expression was measured by FACS. CD4 expression of the transfected GFP expressing cells was analysed using the FlowJo software [software .in vitro studies with proviral HIV subtype B strains have shown that HIV-1 subtype C generally has a lower replication rate [in vivo titers of infected patients, where similar viral loads are observed. We show here that the exchange of the env region of the proviral pMJ4 plasmid enhances infectivity. We have shown recently that the usage of the authentic subtype is required especially for the analysis of resistance mutations, such as a highly mutated proteases, since the viral background is necessary for fitness compensation [HIV-1 subtype C remains the predominate subtype worldwide and is especially prevalent in Sub‑Saharan Africa, where the HIV/AIDS epidemic is highest. However, comparative ion rate ,6. This ensation . The pZA


Giardia lamblia differentiates into infectious cysts within the human intestinal tract for disease transmission. Expression of the cyst wall protein (cwp) genes increases with similar kinetics during encystation. However, little is known how their gene regulation shares common mechanisms. DNA topoisomerases maintain normal topology of genomic DNA. They are necessary for cell proliferation and tissue development as they are involved in transcription, DNA replication, and chromosome condensation. A putative topoisomerase II (topo II) gene has been identified in the G. lamblia genome. We asked whether Topo II could regulate Giardia encystation. We found that Topo II was present in cell nuclei and its gene was up-regulated during encystation. Topo II has typical ATPase and DNA cleavage activity of type II topoisomerases. Mutation analysis revealed that the catalytic important Tyr residue and cleavage domain are important for Topo II function. We used etoposide-mediated topoisomerase immunoprecipitation assays to confirm the binding of Topo II to the cwp promoters in vivo. Interestingly, Topo II overexpression increased the levels of cwp gene expression and cyst formation. Microarray analysis identified up-regulation of cwp and specific vsp genes by Topo II. We also found that the type II topoisomerase inhibitor etoposide has growth inhibition effect on Giardia. Addition of etoposide significantly decreased the levels of cwp gene expression and cyst formation. Our results suggest that Topo II has been functionally conserved during evolution and that Topo II plays important roles in induction of the cwp genes, which is key to Giardia differentiation into cysts.The protozoan  Giardia lamblia becomes infective by differentiation into water-resistant cysts. During encystation, cyst wall proteins (CWPs) are highly synthesized and are targeted to the cyst wall. However, little is known about the regulation mechanisms of these genes. DNA topoisomerases can resolve the topological problems and are needed for a variety of key cellular functions, including cell proliferation, cell differentiation and organ development in higher eukaryotes. We found that giardial Topo II was highly expressed during encystation. Topo II is present in Giardia nuclei and is associated with the encystation-induced cwp gene promoters. Topo II has typical DNA cleavage activity of type II topoisomerases. Interestingly, overexpression of Topo II can induce cwp gene expression and cyst formation. Addition of a type II topoisomerase inhibitor, etoposide, significantly decreased the levels of cwp gene expression and cyst formation. Etoposide also has growth inhibition effect on Giardia. Our results suggest that Topo II plays an important role in induction of encystation by up-regulation of the cwp gene expression. Our results provide insights into the function of Topo II in parasite differentiation into cysts and help develop ways to interrupt the parasite life cycle. Giardia lamblia is an intestinal protozoan parasite causing outbreaks of infectious diarrheal diseases worldwide with an estimated 280 million cases of giardiasis yearly G. lamblia is a valuable model for other intestinal protozoan parasites as its life cycle can be reproduced in vitroG. lamblia also raises great biological interest for understanding the progress of eukaryotic evolution G. lamblia has fewer components of DNA synthesis, transcription, and RNA processing cis-acting elements are sufficient for expression of many genes G. lamblia is transmitted by differentiation into infective cysts, which are protectively walled and can survive in water cwp gene expression have been identified Giardia encystation has been proposed to link to cell cycle regulation and Cdk2 pathway may be involved in activation of Myb2 and up-regulation of cwp genes Topoisomerases can resolve the topological problems of chromosomes in both prokaryotes and eukaryotes Type II topoisomerases create a transient double stranded DNA break by transesterification of a specific Tyr of the enzyme cleavage domain and a phosphodiester bond of DNA Giardia infection, but resistance and side effect limits its use Giardia and other important intestinal protozoan pathogens.DNA topoisomerases are potential therapeutic targets for drug discovery. Many antitumor agents act through inhibiting topoisomerase activity in caner cells G. lamblia genome Giardia cyst nuclei Giardia differentiation into dormant cysts. We found that the expression levels of the Giardia Topo II increased during encystation. In addition, Topo II has typical ATPase, DNA binding, and DNA cleavage activity of type II topoisomerases. We also found that the levels of cyst formation and the cwp1-3 and myb2 gene expression increased by Topo II overexpression, suggesting that Topo II may be an important factor involved in activation of these gene expression and Giardia encystation. We used a method similar to chromatin immunoprecipitation (ChIP) assays, etoposide-mediated topoisomerase immunoprecipitation assays in vivo. We also tested the effect of a type II topoisomerase inhibitor, etoposide, and found that it inhibited Giardia growth and decreased the levels of cyst formation and the cwp1-3 and myb2 gene expression. Because etoposide has side effect Giardia growth but has less side effect. Our results provide insights into the role of type II topoisomerase in inducing Giardia differentiation into dormant cysts and into the development of better drugs for treatment of giardiasis.A putative topoisomerase II (Topo II) has been identified in G. lamblia WB (ATCC 50803), clone C6, were cultured in modified TYI-S33 medium 5 cells/ml.Trophozoites of 6 cells/ml) during vegetative growth as previously described 6 cells/ml. Cells seeded at this density became confluent within 24 h. Confluent cultures were maintained for an additional 8 h to ensure that the cultures were in stationary phase (at a density of ∼2×106 cells/ml). Cyst count was performed on these stationary phase cultures. Cultures were chilled and cells were washed twice in double-distilled water at 4°C and trophozoites were lysed by incubation in double-distilled water overnight at 4°C. Cysts were washed three times in double-distilled water at 4°C. Water-resistant cysts were counted in a hemacytometer chamber. Cyst count was also performed on 24 h encysting cultures.Cyst count was performed on the stationary phase cultures (∼2×10G. lamblia genome database (http://www.giardiadb.org/giardiadb/) NP_001058.2) using the BLAST program XP_001708897.1, open reading frame 16975 in the G. lamblia genome database). The Topo II coding region with 221 bp of 5′- flanking region was cloned and the nucleotide sequence was determined. The topo II gene sequence in the database was correct. To isolate the cDNA of the topo II gene, we performed RT-PCR with topo II-specific primers using total RNA from G. lamblia. For RT-PCR, 5 µg of DNase-treated total RNA from vegetative and 24 h encysting cells was mixed with oligo (dT)12–18 and random hexamers and Superscript II RNase H- reverse transcriptase (Invitrogen). Synthesized cDNA was used as a template in subsequent PCR with primers topo IIF and topo IIR. Oligonucleotides used in this study are listed in The G. lamblia cell line at the differentiation stages indicated in figure legends using TRIzol reagent (Invitrogen). For RT-PCR, 5 µg of DNase-treated total RNA was mixed with oligo (dT)12–18 and random hexamers and Superscript II RNase H− reverse transcriptase (Invitrogen). Synthesized cDNA was used as a template in subsequent PCR. Semi-quantitative RT-PCR analysis of topo II , topo II-ha, cwp1 , cwp2 , cwp3 , myb2 , ran , and 18 S ribosomal RNA  gene expression was performed using primers topo II828F and topo II1311R, topo IIHAF and HAR, cwp1F and cwp1R, cwp2F and cwp2R, cwp3F and cwp3R, myb2F and myb2R, ranF and ranR, 18SrealF and 18SrealR, respectively. For quantitative real-time PCR, SYBR Green PCR master mixture was used (Kapa Biosystems). PCR was performed using an Applied Biosystems PRISMTM 7900 Sequence Detection System (Applied Biosystems). Specific primers were designed for detection of the topo II, topo II-ha, cwp1, cwp2, cwp3, myb2, ran, and 18 S ribosomal RNA genes: topo IIrealF and topo IIrealR; topo IIHAF and HAR; cwp1realF and cwp1realR; cwp2realF and cwp2realR; cwp3realF and cwp3realR; myb2realF and myb2realR; ranrealF and ranrealR; 18SrealF and 18SrealR. Two independently generated stably transfected lines were made from each construct and each of these cell lines was assayed three separate times. The results are expressed as relative expression level over control. Student's t-tests were used to determine statistical significance of differences between samples.Total RNA was extracted from topo II gene and its 221 bp of 5′- flanking region were amplified with oligonucleotides topo IIKF and topo IIAR, digested with KpnI and AvrII, and cloned into KpnI and XbaI digested pPop2N topo II gene was amplified using two primer pairs topo IIm1F and topo IIAR, and topo IIm1R and topo IIKF. The two PCR products were purified and used as templates for a second PCR. The second PCR also included primers topo IIKF and topo IIAR, and the product was digested with KpnI and AvrII and cloned into the KpnI and XbaI digested pPop2N. To make construct pPTopo IIm2 or pPTopo IIm3, the topo II gene was amplified using primers topo IIKF and topo IIm2AR or topo IIm3AR, digested with KpnI and AvrII, and cloned into KpnI and XbaI digested pPop2N topo II gene was amplified with primers topo II5XF and topo II5NR, digested with XbaI and NcoI, and ligated in place of the NheI/NcoI-excised ran promoter sequence in pPop2N. To make construct pPTopo II5m, the 300-bp 5′-flanking region of the topo II gene was amplified with primers topo II5XF and topo II5mNR, digested with XbaI and NcoI, and ligated in place of the NheI/NcoI-excised ran promoter sequence in pPop2N.All constructs were verified by DNA sequencing with a BigDye Terminator 3.1 DNA Sequencing kit and an Applied Biosystems 3100 DNA Analyser (Applied Biosystems). Plasmid 5′Δ5N-Pac was a gift from Dr. Steven Singer and Dr. Theodore Nash pac gene were selected and maintained with 54 µg/ml of puromycin as described 6 cells/ml) or 24 h encysting cells as described Cells transfected with the pP series plasmids containing the topo II gene was amplified using oligonucleotides topo IIF and topo IIR. The product was cloned into the expression vector pET101/D-TOPO (Invitrogen) in frame with the C-terminal His and V5 tag to generate plasmid pTopo II. To make pTopo IIN expression vector, the topo II gene was amplified using primers topo IIF and topo IINR and cloned into the expression vector. To make pTopo IIC expression vector, the topo II gene was amplified using primers topo IICF and topo IIR and cloned into the expression vector. To make the pTopo IICm1, pTopo IICm2, or pTopo IICm3 expression vector, the topo II gene was amplified using primers topo IICF and topo IIR and specific template, including pPTopo IIm1, pPTopo IIm2, or pPTopo IIm3, and cloned into the expression vector. The pTopo IIN, pTopo IIC, pTopo IICm1, pTopo IICm2, or pTopo IICm3 plasmid was freshly transformed into Escherichia coli BL21 Star (DE3) (Invitrogen). An overnight pre-culture was used to start a 250-ml culture. E. coli cells were grown to an A600 of 0.5, and then induced with 1 mM isopropyl-D-thiogalactopyranoside (IPTG) (Promega) for 4 h. Bacteria were harvested by centrifugation and sonicated in 10 ml of buffer A  containing 10 mM imidazole and protease inhibitor mixture (Sigma). The samples were centrifuged, and the supernatant was mixed with 1 ml of 50% slurry of nickel-nitrilotriacetic acid Superflow (Qiagen). The resin was washed with buffer A containing 20 mM imidazole and eluted with buffer A containing 250 mM imidazole. Fractions containing Topo II, Topo IIN, Topo IIC, Topo IICm1, Topo IICm2, or Topo IICm3 were pooled, dialyzed in 25 mM HEPES pH 7.9, 20 mM KCl, and 15% glycerol, and stored at −70°C. Protein purity and concentration were estimated by Coomassie Blue and silver staining compared with serum albumin. Topo II, Topo IIN, Topo IIC, Topo IICm1, Topo IICm2, or Topo IICm3 was purified to apparent homogeneity (>95%).The genomic Purified Topo II protein was used to generate rabbit polyclonal antibodies through a commercial vendor .6 cells/coverslip) and then fixed and stained The pPTopo II, pPTopo IIm1, pPTopo IIm2, or pPTopo IIm3 stable transfectants were cultured in growth medium under puromycin selection. Cells cultured in growth medium or encystation medium for 24 h were harvested, washed in phosphate-buffered saline (PBS), and attached to glass coverslips  for 96 well plate and 200 µl sample = 0.56). Lineweaver-Burk plot was used to determine Km and Vmax.ATPase assays were performed using purified Topo II and pyruvate kinase/lactate dehydrogenase reaction as described Cleavage assays were performed as described www.topogen.com). Reaction was performed in a 20 ul mixture containing 40 mM Tris-HCl pH 7.8, 100 mM KCl, 18 mM MgCl2, 0.5 mM DTT, 0.5 mM EDTA, 1 mM ATP, 30 ug/ml BSA, 100 ng kDNA, and 40 ng purified TopoII. Various topoisomerase inhibitors were also added to the reactions to test the effect on decatenation activity of topoisomerases. After incubation at 37oC for 30 min, reaction was stopped by addition of 0.5% SDS, 10 mM EDTA, and 2 mg proteinase K and incubation at 37oC for 30 min. The resulting DNA was separated by electrophoresis on 1% agarose gels with ethidium bromide.Decatenation assays were performed as described (7 cells in 45 ml medium) and harvested after 24 h in encystation medium and washed in phosphate-buffered saline. ChIP was performed as described previously topo II, cwp1, cwp2, cwp3, myb2, and ran gene promoters within the −200 to −1 region.The WB clone C6 cells were inoculated into encystation medium (5×107 cells in 45 ml medium) and harvested after 24 h and washed in phosphate-buffered saline. The assay was performed as described previously topo II, cwp1, cwp2, cwp3, myb2, and ran gene promoters within the −200 to −1 region.The WB clone C6 cells were inoculated into encystation medium containing 400 µM etoposide  at 65°C for 17 h. After washing and drying by nitrogen gun blowing, microarrays were scanned with an Agilent microarray scanner (Agilent Technologies) at 535 nm for Cy3 and 625 nm for Cy5. Scanned images were analyzed by Feature Extraction version 10.5.1.1 software (Agilent Technologies), and image analysis and normalization software was used to quantify signal and background intensity for each feature; data were substantially normalized by the rank consistency filtering LOWESS method. All data is MIAME compliant and that the raw data has been deposited in a MIAME (http://www.mged.org/Workgroups/MIAME/miame.html) compliant database (GEO) with accession number GSE39665.RNA was quantified by A260 nm by an ND-1000 spectrophotometer  and qualitated by a Bioanalyzer 2100 (Agilent Technology) with an RNA 6000 Nano LabChip kit. RNA from the pPTopo II cell line was labeled by Cy5 and RNA from the 5′▵5N-Pac cell line was labeled by Cy3. In another experiment, RNA from the etoposide treated cells was labeled by Cy5 and RNA from non treated cells was labeled by Cy3. 0.5 µg of total RNA was amplified by a Low RNA Input Quick-Amp labeling kit (Agilent Technologies) and labeled with Cy3 or Cy5  during the G. lamblia, we searched the G. lamblia genome database (http://giardiadb.org/giardiadb/) XP_001708897.1, open reading frame 16975 in the G. lamblia genome database). Two of them are similar to type IA topoisomerases and one is similar to spo11 Type II DNA topoisomerases. We first focused on understanding the role of topoisomerase II (Topo II) in Giardia. This Topo II has been reported previously topo II gene contained no introns. The deduced Giardia Topo II protein contains 1491 amino acids with a predicted molecular mass of ∼164.01 kDa and a pI of 8.47. It has one putative DNA gyrase B domain (residues 337 to 436) and two domains of DNA gyrase/topoisomerase IV, A subunit (Topo IV domains) (residues 755 to 997 and 1057 to 1322) as predicted by Pfam (http://pfam.sanger.ac.uk/) Giardia Topo II (residues 52 to 201) and a conserved G-loop motif (GXXGXGXX) for ATP binding is also present in Giardia Topo II  (Giardia Topo II is moderately similar to the human topoisomerase IIα/β and the size of Giardia Topo II (1491 amino acids) is smaller than that of human topoisomerase IIα/β (1531/1621 amino acids) , corresponding to the catalytic important Tyr of human topoisomerase IIα and IIβ  (To identify genes encoding novel topoisomerase proteins from  by Pfam  and S1(hRNGYGAK)  and S1 [o acids) . Giardiactively)  [67].Giardia Topo II (Giardia Topo II has 28% (27%) identity and 41% (39%) similarity to that of human topoisomerase IIα/β  have 30% identity and 45% similarity to the Topo IV domain of the human topoisomerase IIα/IIβ (residues 713–1171 or 729–1184)  relative to the 5′Δ5N-Pac control cell line (cwp1-3 and myb2 genes in the Topo II overexpressing cell line increased by ∼3.32 to ∼10.24-fold (p<0.05) relative to the 5′Δ5N-Pac control cell line . Similar results were obtained during encystation (cwp1-3 and myb2 genes.To study the role of Topo II in ell line . The mRNell line . Similarystation . Our resG. lamblia trophozoites may undergo spontaneous differentiation G. lamblia cultures during growth to stationary phase (∼4800 cysts/ml for 5′Δ5N-Pac cell line) p<0.05) relative to the control cell line, indicating that the overexpressed Topo II can increase cyst formation   . We alsope value). Similardetected . We alsodetected . The levdetected . Similardetected . The rescwp1, cwp2, and myb2 expression in the Topo II overexpressing cell line ∼1.75 to ∼3.15-fold of the levels in the control cell line . Similar mRNA levels of the ran gene were detected (vsp genes in the Topo II overexpressing cell line (p<0.05) in the Topo II overexpressing cell line relative to the vector control, respectively (topo II gene in the Topo II overexpressing cell line increased by ∼2.27-fold (p<0.05)(Oligonucleotide microarray assays confirmed up-regulation of ell line . Expressdetected . Oligonuell line . We founectively . Express (p<0.05).E. coli and purified it to >95% homogeneity, as assessed in a silver-stained gel  We further tested DNA-binding activity of Topo II. Electrophoretic mobility shift assays were performed with the purified Topo II protein and double-stranded DNA sequences from the 5′-flanking region of cwp1 promoter (cwp1-45/-1) (cwp1 gene. Incubation of the GC rich probes 18S-30/-1 and 18S-60/-31 (Giardia Topo II can bind the cwp1 promoter (−45/−1 region).Topo IIC was also shown to bind to the 1-45/-1) , lane 2.S-60/-31  with TopS-60/-31 . The forS-60/-31 , lane 8.Competition analysis was used to test the binding affinity of DNA fragments for Topo IIC. DNA fragments with the higher binding affinity for Topo IIC are more effective in the competition analysis. Topo IIC was also shown to bind weakly to the cwp1-90/-46 , lane 3.To investigate how Topo IIC binds DNA, we used distamycin A, which binds to the DNA minor groove, as a competitive inhibitor of Topo IIC binding cwp1, cwp2, cwp3, myb2, and ran promoters during encystation (We further used etoposide-mediated topoisomerase immunoprecipitation assay ystation . Howeverystation (data notcwp1-3 genes and its own gene by binding to specific sequences cwp1 promoter topo II gene. We expressed Myb2 in E. coli and purified it to >95% homogeneity to test the DNA binding activity of Myb2 (data not shown). Electrophoretic mobility shift assays were performed with the purified Myb2 protein and double-stranded DNA sequences from the 5′-flanking region of cwp1 and topo II genes. Incubation of a labeled double-stranded DNA probes cwp1-90/-46 with Myb2 resulted in the formation of retarded bands . As a control, the 18 S ribosomal RNA levels did not change in the Myb2 overexpressing cell line compared with the control cell line  , resulted in a significant decrease of luciferase activity to ∼71% and ∼54% of the wild-type value in vegetative and encysting cells, respectively, and a slight decrease in the induction to ∼1.93 (topo II promoter function as a positive cis-acting element in both vegetative and encysting stages.We further investigated the ability of the Myb2 binding site to regulate the o ∼2.53) . Mutatioto ∼1.93 . These rGiardia and etoposide can trap cleavage complex of Topo II. We also found that etoposide has a significant anti-Giardia effect   . We alsoe points . Within cells/ml .topo II, cwp1, cwp2, cwp3, and myb2 genes by ∼0.61, ∼0.60, ∼0.51, ∼0.45, and ∼0.56-fold (p<0.05) . Similar mRNA levels of the ran gene were detected (data not shown). We found that 56 and 48 genes were significantly up-regulated (>2-fold) and down-regulated (<1/2)(p<0.05) in the etoposide treated cells relative to the control cells, respectively  and one in mitochondria (TbTOP2mt) Type II topoisomerases are required for cell proliferation, tissue development, and cell cycle progression in higher eukaryotes Giardia differentiation into dormant cysts Giardia Topo II may help encystation. Our results show that the giardial Topo II localizes to the cell nuclei and an increase of the Topo II protein during encystation  Giardia Topo II functions more like the eukaryotic type II topoisomerases.Although divergent from human topoisomerase IIα/β proteins, the full-length activity –6. This Giardia Topo II  of Giardia Topo II is near the C terminus  (http://psort.nibb.ac.jp/), including PKTKRTK at 295, RKVLYACFKRNLKTKLK at 769, PSRKHRI at 1200, PKPKKEH at 1395, KPKK at 1396, PTEPKRK at 1424, PKRKRPA at 1427, PKRK at 1427, KRKR at 1428, RKRP at 1429. Deletion of the C-terminal 153 amino acids (residues 1338 to 1491) (pPTopo IIm2) (cwp1-3 and myb2 mRNA (Topo IIm2)    and 4. So IICm1)  resultedpo IIm2)  resultedpo IIm2) , suggestpo IIm2)  and S5A,po IIm2) , suggest nucleus  and S5A. nucleus  resultedo IICm3)  and 4. TGiardia Topo II . We also found that addition of etoposide decreased the topo II promoter activity  in the Topo II overexpressing cell line relative to the vector control using oligonucleotide microarray assays (p<0.05) in the etoposide treated cells relative to the control cells, respectively (Giardia. Seven genes listed in We found that 95 and 20 genes were significantly up-regulated (>2-fold) and down-regulated (<1/2)Click here for additional data file.Figure S2Alignment of the Topo IV domains of the Topo II proteins.(PDF)Click here for additional data file.Figure S3Phylogenetic analysis of Topo II proteins.(PDF)Click here for additional data file.Figure S4cwp1-3 and myb2 gene expression in the Topo II overexpressing cell line during encystation.Induction of (PDF)Click here for additional data file.Figure S5Analysis of Topo II function.(PDF)Click here for additional data file.Figure S6vsp promoter.Binding of Topo IIC to (PDF)Click here for additional data file.Table S1Oligonucleotides used in this study.(PDF)Click here for additional data file.Table S2Genes up or down regulated by Topo II overexpression(PDF)Click here for additional data file.Table S3Genes up or down regulated by etoposide treatment(PDF)Click here for additional data file.