Genet. Genomics42, 477–485. (doi:10.1016/j.jgg.2015.07.004); Combelles et al. 2011 Hum. Reprod.26, 545–552. (doi:10.1093/humrep/deq363); Escrich et al. 2011 J. Assist. Reprod. Genet.28, 111–117. (doi:10.1007/s10815-010-9493-5)). However, the mechanisms that control the meiotic arrest in mammalian eggs are only poorly understood. Here, we report that a complex of BTG4 and CAF1 safeguards metaphase II arrest in mammalian eggs by deadenylating maternal mRNAs. As a follow-up of our recent high content RNAi screen for meiotic genes ), we identified Btg4 as an essential regulator of metaphase II arrest. Btg4-depleted eggs progress into anaphase II spontaneously before fertilization. BTG4 prevents the progression into anaphase by ensuring that the anaphase-promoting complex/cyclosome (APC/C) is completely inhibited during the arrest. The inhibition of the APC/C relies on EMI2 ; Ohe et al. 2010 Mol. Biol. Cell21, 905–913. (doi:10.1091/mbc.E09-11-0974)), whose expression is perturbed in the absence of BTG4. BTG4 controls protein expression during metaphase II arrest by forming a complex with the CAF1 deadenylase and we hypothesize that this complex is recruited to the mRNA via interactions between BTG4 and poly(A)-binding proteins. The BTG4–CAF1 complex drives the shortening of the poly(A) tails of a large number of transcripts at the MI–MII transition, and this wave of deadenylation is essential for the arrest in metaphase II. These findings establish a BTG4-dependent pathway for controlling poly(A) tail length during meiosis and identify an unexpected role for mRNA deadenylation in preventing the spontaneous activation of eggs.Once every menstrual cycle, eggs are ovulated into the oviduct where they await fertilization. The ovulated eggs are arrested in metaphase of the second meiotic division, and only complete meiosis upon fertilization. It is crucial that the maintenance of metaphase arrest is tightly controlled, because the spontaneous activation of the egg would preclude the development of a viable embryo (Zhang  Btg4-depleted oocytes had no apparent defects during the first meiotic division (a): they underwent nuclear envelope breakdown (NEBD) and anaphase I with similar efficiency and timing as oocytes microinjected with control siRNAs . In addition, the formation of metaphase II (MII) spindles was not affected (a). However, 63% of Btg4-depleted oocytes spontaneously progressed into anaphase II within 5.4 ± 1.7 h after anaphase I, whereas the control oocytes remained arrested in metaphase . This precocious release from metaphase II arrest was accompanied by a high incidence of lagging chromosomes . Under physiological conditions, the release from metaphase II arrest is triggered by fertilization and results in meiotic exit and the formation of a pronucleus [Btg4-depleted oocytes that underwent anaphase II proceeded directly into a non-physiological third meiotic division (MIII) and formed a distorted MIII spindle that fragmented and had misaligned chromosomes (figure 1a). This phenotype was specifically due to the depletion of Btg4, because it could be rescued by the expression of EGFP-tagged BTG4 (figure 1d).We recently carried out a high content RNAi screen to identify new meiotic genes in mammalian oocytes . In the division a: they uaffected a. Howeveonucleus –6. The B2.Btg4-depleted oocytes was due to impaired inhibition of the APC/C by monitoring the levels of APC/C activity in live oocytes throughout maturation. To this end, we expressed fluorescent cyclin B and securin in oocytes undergoing meiosis and quantified their intensity. As expected, cyclin B and securin levels were high after NEBD and both proteins were rapidly degraded before the onset of anaphase I, when the APC/C was activated (a). In control oocytes, the levels of cyclin B and securin were partially restored after the extrusion of the first polar body, when the metaphase II spindle was formed (a), indicative of effective inhibition of the APC/C. In contrast, Btg4-depleted oocytes failed to re-accumulate securin and cyclin B after the completion of the first meiotic division, indicating that the APC/C was not efficiently inhibited. Residual inhibition of the APC/C was probably achieved, because the levels of cyclin B and securin still slowly increased following anaphase I, albeit at a much lower rate than in control oocytes. The cyclin B and securin levels decreased again when the oocytes progressed into anaphase II . The APC/C is activated at the metaphase-to-anaphase transition during the first meiotic division, when it triggers the progression into anaphase by targeting cyclin B and securin for degradation. Subsequently, it becomes inactivated and inhibited during metaphase II arrest, so that the levels of cyclin B and securin remain high –10. We tphase II .Figure Two major pathways are known to mediate metaphase II arrest and the inhibition of the APC/C ,11. The Emi2-depleted oocytes enter interphase and form a pronucleus after completion of the first meiotic division [a,e). The second pathway involves the MOS/MEK/MAPK signalling cascade and is required for the maintenance of metaphase II arrest [Mos-depleted oocytes undergo two consecutive meiotic divisions and proceed to metaphase III, similarly to Btg4-depleted oocytes . However, in contrast to Btg4-depleted oocytes, they extrude abnormally large polar bodies and transiently form a nucleus before entering second meiotic division . The exact mechanism by which the MOS/MEK/MAPK pathway mediates metaphase II arrest in mammalian oocytes remains to be determined. Given the essential function of the EMI2- and MOS-dependent pathways in metaphase II arrest, we tested if the activity of these pathways is affected upon Btg4 depletion.EMI2 is expressed only when the oocytes have progressed into anaphase I and is required for both the establishment and the maintenance of metaphase II arrest, and division ,14 . In contrast, Emi2-depleted oocytes were unable to inhibit the APC/C as evident from the complete degradation of cyclin B and securin (a). The inhibition of the APC/C was thus severely impaired in both Emi2- and Btg4-depleted oocytes, with slightly lower levels of APC/C activity in Btg4-depleted oocytes.First, we compared the degree of APC/C inhibition in Btg4 depletion. RNAi against Btg4 did not perturb MAPK activation during metaphase II as evident from the level of phosphorylated and thus active MAPK . In contrast, Btg4 depletion did indeed impair the EMI2-dependent pathway: western blots with an EMI2-specific antibody revealed that the levels of EMI2 were strongly decreased upon Btg4 depletion . The impaired inhibition of the APC/C in the absence of BTG4 as well as the release from metaphase II arrest could thus be facilitated by the decrease in EMI2 levels.We then tested directly if either the MOS/MEK/MAPK or EMI2 pathways were affected upon 3.d). A recent study reported a similar pattern of cellular localization, yet showed that the endogenous protein is synthesized only in metaphase II [Btg4 depletion . In addition, co-depletion of Cnot7 and Cnot8 caused chromosome alignment defects, lagging chromosomes during anaphase I and an increase in the fraction of oocytes that arrested in metaphase I . This wide range of defects is likely to reflect the importance of the CCR4–NOT complex in several steps of meiosis and indicates that it has a broad spectrum of targets [figure 3c). Importantly, the mutant BTG4 was unable to rescue metaphase II arrest in Btg4-depleted oocytes (figure 3d). This shows that BTG4's function during oocyte maturation crucially depends on its interaction with CNOT7 and CNOT8, and suggests that BTG4 prevents the spontaneous resumption of meiosis by controlling mRNA deadenylation.The decrease in EMI2 levels upon BTG4 depletion suggested a function of BTG4 in controlling gene expression. Consistent with such a function, exogenous EGFP-tagged BTG4 localized to both the nucleus and the cytoplasm of a maturing oocyte, where it could be involved in controlling gene expression  or caused a metaphase I arrest , reminiscent of the phenotypes observed upon Btg4 or Cnot7/Cnot8 depletion. The model that poly(A)-binding proteins such as PABPN1 recruit the BTG4/CCR4–NOT complex to its target mRNAs is further supported by the fact that other members of the BTG/TOB family also interact with poly(A)-binding proteins [in vivo.BTG4 does not contain any known RNA-binding domain. It is thus likely to rely on a second interaction partner to mediate binding of the CCR4–NOT complex to mRNA. A recent study suggested that BTG4 may interact with eIF4E to bind to mRNAs . Interesproteins . Recentlproteins . This su4.Btg4-depleted early MII oocytes (before the release from MII arrest) and compared the transcriptomes by RNA sequencing (RNA-seq). This revealed prominent differences in transcript abundancies in the poly(A)-enriched fraction. In particular, a large number of transcripts were upregulated in Btg4-depleted oocytes . The increase in the fraction of poly(A) tailed mRNAs must have resulted from impaired mRNA deadenylation or degradation, because oocytes at this developmental stage do not synthesize new RNA [Btg4−/− mice [−/−Btg4 mice, the timely degradation of mRNA at MI–MII transition is perturbed and that several tested mRNAs that should have been deadenylated at this transition, such as Padi6, Zp2 and Fgf2, retained their poly(A) tails. However, what is the link between impaired mRNA deadenylation in Btg4-depleted oocytes and the release from metaphase II arrest? We observed that the levels of EMI2 were decreased upon Btg4 depletion. Interestingly, the reduction of EMI2 was not due to a decrease in the amount of polyadenylated Emi2 mRNA . We hypothesized that the observed phenotypes could instead be caused by the presence of excess amounts of polyadenylated mRNA in the absence of BTG4. Transcripts that should normally be deadenylated or degraded during the MI–MII transition may occupy the translational machinery, for instance by sequestering poly(A) binding proteins. As a result, this could lead to an insufficient capacity of the oocyte to translate the mRNAs that are essential for metaphase II arrest, such as mRNAs encoding EMI2. To test this hypothesis, we microinjected identical amounts of mRNA encoding EGFP into control and Btg4-depleted oocytes in metaphase II. We observed that the expression of EGFP was decreased by 79% in Btg4-depleted oocytes (figure 4c). We also microinjected oocytes in prophase with mRNA encoding EGFP under the control of the 3′UTR of cyclin A2, which is known to be partly degraded during meiosis I and re-synthesized during metaphase II arrest [figure 4d). We observed that the expression was decreased in Btg4-depleted oocytes by about 55% in comparison with the control group (figure 4d). To test if this reduction results from saturation of the translational machinery or overall inhibition of translation, we compared the translation rates in control and Btg4-depleted MII oocytes. To this end, we incubated oocytes that had completed the first meiotic division in medium containing l-homopropargylglycine (HPG), an analogue of methionine that can be detected using Click-IT chemistry (Life Technologies). The HPG signal that is indicative of the overall level of translation was nearly three times higher in Btg4-depleted oocytes than in controls , suggesting that global translation is increased upon Btg4 depletion. In further support of our hypothesis that the abundancy of polyadenylated mRNAs is responsible for the phenotype of Btg4 depletion, microinjection of in vitro polyadenylated EGFP mRNA into metaphase II arrested oocytes triggered the onset of anaphase II, whereas microinjection of EGFP mRNA without the poly(A) tail had no effect . Collectively, these data show that Btg4 depletion causes an increase in polyadenylated mRNAs and reduced expression of proteins that are normally expressed during metaphase II arrest. An increase in the abundancy of polyadenylated mRNAs and the resulting imbalance in protein expression are sufficient to trigger the spontaneous resumption of meiosis.To test for a role of BTG4 in mRNA deadenylation, we constructed cDNA libraries from mRNAs containing poly(A) tails in control and  new RNA ,29. Cons−/− mice . The stuI arrest . The 3′UI arrest ,31,32. A5.in vivo analysis of the function of BTG4 sheds light on general mechanisms underlying metaphase II arrest in mammalian oocytes, a stage of critical importance for fertilization and the development of a viable embryo [Our e embryo ,11. The e embryo . We prope embryo ,34, yet Btg4−/− mice have been reported to be infertile but otherwise viable [Btg4 a good candidate whose mutation could contribute to the spontaneous abortive activation of eggs that has been implicated in infertility in humans [Btg4 in the formation of a viable egg and suggest that the impaired embryonic development and female infertility observed in −/−Btg4 mice [−/−Btg4 mutant mice also affects the stages after fertilization and thereby contributes to defects during embryo development. Interestingly, mutations in other BTG/TOB genes have also been linked to several types of cancer [The poly- and deadenylation of mRNAs is a major mechanism of regulating gene expression in oocytes ,31, yet e viable . This sun humans ,36. Our −/− mice  originatf cancer –40. The 6.6.1.1 females were isolated, microinjected and cultured as previously described [All animals were maintained in a specific pathogen-free environment according to UK Home Office regulations. Fully grown oocytes from FVB females or follicle-enclosed oocytes from (C57BL × CBA) FBTG4AAGGGCTTGTGCTGAGAGTAAAAGCCTGAATATTTGACTCTAAAGGAAGACTTGAAATAAATACNOT7AGCAAGCATTCTGTTATGAAACAGGAAGTTGCTGAGCAGTTACTGGATGAAGAGATGAAGAAACNOT8AAGGACAATTGTAAGAAGTAAATGCTTGTAAATATTATTTATTGCAATGTTGATCTTCTTAAATTGCTCTGAATTTGTAAACAAEMI2CGGCATGAACTTTAAAGGCAAAAACTTCTAAATGATGATTTATCCATCAAATGTAATCTATAAAAGCAGATCTCTGGAAAGTAAMOSCCCGAAGACTCCAACAGCCTAAACAGGTATGTCTGATGCATACTCGGTGTATAAAGCCACTTACACGCCCAAAGCTGACATCTAPABPN1CCCAAAGGGTTTGCATATATACCGGAGCTAGAAGCGATCAAACCCAGTGATCATGTCTCTTGAAGGAAGAGGCTGAGAAGCTAA6.2.Mus musculus Btg4 and cyclin B ORFs as well as Emi2 and cyclin A2 3′UTR sequences were amplified from mouse oocyte cDNA. The Btg4 ORF was inserted into pmEGFP-C1 and pmEGFP-N1 vectors (Clontech), using EcoRI and BamHI. EGFP–Btg4 and Btg4–EGFP were then subcloned into pGEMHE, using AgeI and BamHI or NheI and NotI, respectively. pGEMHE–EGFP–Btg4 was mutated using the QuickChange II XL site-directed mutagenesis kit (Agilent). The cyclin B ORF was cloned into the pmCherry-N1 vector (Clontech), using AgeI and NheI. Cyclin B-mCherry was then subcloned into the pGEMHE vector, using NheI and NotI. To obtain pGEMHE–securin–mCherry, the Mus musculus securin ORF was cloned into the pGEMHE vector, using EcoRI and BamHI; and mCherry was sublconed into the same vector, using BamHI and NotI. The Mus musculus Cnot7 or Cnot8 ORFs were ligated using the Gibson assembly kit (NEB) with pFlag-C3 that was linearized with XhoI. pT7-mEGFP-Emi2 3′UTR was created by ligating EGFP and the 3′ UTR of mouse Emi2 by Gibson assembly and subcloning into a modified pmEGFP-N1 vector with added T7. For mRNA synthesis, the pGEMHE constructs were linearized with AscI. pT7-mEGFP-Emi2 3′UTR was linearized with NotI. Capped mRNA was synthesized using T7 polymerase , following the manufacturer's instructions. Final mRNA concentrations were determined on ethidium bromide agarose gels by comparison of the total band intensity with an RNA standard (Ambion). When stated, mRNA tailing was performed using Poly(A) tailing kit (Ambion).Previously published constructs were used for the visualization of chromosomes, pGEMHE-H2B–mRFP1 , or meioCapped mRNA was synthesized using T7 polymerase , following the manufacturer's instructions. Final mRNA concentrations were determined on ethidium bromide agarose gels by comparison of the total band intensity with an RNA standard (Ambion).6.3.Btg4-depleted oocytes were incubated in M2 medium supplemented with 50 µM HPG for 2 h. The oocytes were fixed for 30 min at 37°C in 100 mM HEPES , 50 mM EGTA , 10 mM MgSO4, 2% formaldehyde (methanol free) and 0.2% Triton X-100, based on previously published methods. Oocytes were permeabilized in PBS with 0.5% Triton X-100 for 30 min at room temperature and washed in PBS + 3% BSA. HPG was detected, using Click-iT cell reaction kit (Life Technologies). The mean cytoplasmic signal was measured in the middle section of each oocyte.MII or equivalent control and 6.4.en (Zeiss).All images were acquired with a Zeiss LSM710 confocal microscope equipped with a Zeiss environmental incubator box or a Zeiss LSM780 confocal microscope equipped with a Tokai hit stage top incubator, with a C-Apochromat 40×/1.2 W water immersion objective lens. In most of the images, shot noise was reduced with a Gaussian filter, and maximum intensity z-projections are shown. The z-projections were generated in Z6.5.The mean cytoplasmic signal of mCherry-tagged cyclin B or securin was measured in a selected area in the middle section of an oocyte over the entire course of meiosis, and the mean background signal from outside the boundaries of the oocyte was subtracted. The signal was then normalized to the starting intensity . The measurements were aligned to anaphase onset in each oocyte (time 0). For EGFP measurements, nominal fluorescence values were compared and plotted over time from the start of the imaging.6.6.t-test or Fisher's exact test (two-tailed) were calculated in Microsoft Excel. No statistical methods were used to predetermine sample size. All error bars show standard deviation. All box plots show median (line), mean , 5th, 95th (whiskers) and 25th and 75th percentile (boxes).Mean, s.d. and statistical significance based on Student's 6.7.mRNA was extracted from oocytes (10–30 per sample), using an RNeasy mini kit (Qiagen) for total RNA or Dynabeads mRNA DIRECT micro kit (Ambion) for the polyadenylated mRNA. The cDNA was generated, using a high-capacity RNA-to-cDNA kit (Applied Biosystems). Real-time PCR was performed with the 7900 HT real-time fast PCR system (Applied Biosystems), using SYBR Green as readout. GAPDH mRNA was used for normalization as the reference gene.6.8.2. Cells were transfected using polyethylenimine. Proteins were immunoprecipitated using the magnetic GFP-Trap kit (ChromoTek) and dissociated from the beads by boiling in sample buffer. Immunoprecipitates were resolved on protein gels for western blot.HeLa cells were cultured in DMEM GlutaMAX medium (Gibco) at 37°C in 10% CO6.9.The Y2H screen was performed by Hybrigenics, using full-length EGFP–BTG4 as a bait and a mouse ovarian cDNA library.6.10.Total RNA was isolated, using NucleoSpin RNA XS (Macherey-Nagel) from 50 RNAi-treated oocytes at early metaphase II per sample. A cDNA library was prepared using SMARTer UltraLow Input RNA for sequencing (Clontech Laboratories), and the samples were processed by BGI Tech Solutions. The cDNA product was synthesized and amplified using a SMARTer PCR cDNA synthesis kit (Clontech Laboratories) from the total RNA (about 10 ng) of each sample. The cDNA was fragmented by Covaris E210, and the median insert length was about 200 base pairs. The paired-end cDNA library was prepared in accordance with Illumina's protocols with an insert size of 200 base pairs and sequenced for 100 base pairs by HiSeq4000 (Illumina). The differential gene expression was assessed using NOISeq and DESeq2, as described in the electronic supplementary material.6.11.Btg4-depleted oocytes and the control group, we used a non-parametric method encoded in NOISeq and a parametric method DESeq2. For NOISeq, we first filtered for low count or abundance using the ‘CPM’ low count filter of NOISeq. Significant differential expression between groups was determined using NOISeq with the following parameters: (i) ‘tmm’, trimmed mean of log2 FPKM, normalization; (ii) biological replicates data; and (iii) probability of differential expression q being set to 0.8 or above and log2 values being greater than or equal to −1 or 1 for upregulated and downregulated genes, respectively, in Btg4-depleted oocytes. This yielded 149 upregulated and 12 downregulated genes in Btg4-depleted oocytes. Further, the DESeq2 standard pipeline for analyses was followed as described [RNA-Seq-based measurements of transcript abundances at the level of genes were represented by fragments per kilobase of transcript per million fragments mapped (FPKMs). FPKM is conceptually similar to the reads per kilobase per million reads sequenced (RPKM) measure, but it is easily adaptable for sequencing data from one to higher numbers of reads from single source molecules. To identify significantly differentially expressed genes between escribed , which y6.12.For western blot, 25–50 oocytes per sample were collected. Alternatively, cell lysates (input) or protein immunoprecipitates were collected as described above. Samples were heated at 95°C for 5 min with 1× sample buffer  with NuPage antioxidant (Invitrogen). Proteins were separated on 4–12% gradient NuPage bis–tris precast gels (Invitrogen) in MES–SDS running buffer (Formedium). Proteins were blotted onto nitrocellulose membranes (Amersham) at a constant current of 400 mA for 1 h. Membranes were blocked in 5% skimmed milk in PBST for 1 h at room temperature and incubated with primary antibodies  at 4°C overnight with secondary antibodies  for 1–2 h at room temperature. The detection was performed, using ECL Prime or ECL Advanced (Amersham) and Kodak X-Ray films.


Cassava Vein Mosaic Virus  and identified CsVMV8 (-215 to +166) as the highest expressing fragment in both transient and transgenic assays. Further, we designed a new chimeric promoter ‘MUASCsV8CP’ through inter-molecular hybridization among the upstream activation sequence (UAS) of Mirabilis Mosaic Virus  and CsVMV8, as the core promoter (CP). The MUASCsV8CP was found to be ∼2.2 and ∼2.4 times stronger than the CsVMV8 and CaMV35S promoters, respectively, while its activity was found to be equivalent to that of the CaMV35S2 promoter. Furthermore, we generated transgenic tobacco plants expressing the totiviral ‘Killer protein KP4’ (KP4) under the control of the MUASCsV8CP promoter. Recombinant KP4 was found to accumulate both in the cytoplasm and apoplast of plant cells. The agar-based killing zone assays revealed enhanced resistance of plant-derived KP4 against two deuteromycetous foliar pathogenic fungi viz. Alternaria alternata and Phoma exigua var. exigua. Also, transgenic plants expressing KP4 inhibited the growth progression of these fungi and conferred significant fungal resistance in detached-leaf and whole plant assays. Taken together, we establish the potential of engineering “in-built” fungal stress-tolerance in plants by expressing KP4 under a novel chimeric caulimoviral promoter in a transgenic approach.Development of disease-resistant plant varieties achieved by engineering anti-microbial transgenes under the control of strong promoters can suffice the inhibition of pathogen growth and simultaneously ensure enhanced crop production. For evaluating the prospect of such strong promoters, we comprehensively characterized the full-length transcript promoter of  Colletotrichum graminicola causing anthracnose of turf grasses    in Finlaicus L.) .Till date there have been numerous advances toward the development of disease-resistant plant varieties via conventional breeding . Howevercis-architecture usually appear to be efficient in driving transgenes in a plant cell (Mirabilis Mosaic Virus (MMV) is capable of driving constitutive expression of heterologous genes in engineered plants such as, the SAC domain of PAR-4 (Prostate Apoptosis Response 4) (Glucocerebrosidase (GCB) (hβD-1 and hβD-2) in transgenic tobacco lines (Cassava vein mosaic virus (CsVMV), as CP and tested its efficacy in plant modification. The CsVMV, a putative member of the caulimoviral group infects cassava (Manihot esculenta L.) plants in Brazil. It has a unique genome organization with a promoter sequence that contains several organ-specific cis- elements and has been partially characterized in rice and cassava (The need to develop proficient plant-expression systems coupled to novel regulatory elements (promoter) that can efficiently escalate the disease resistance by expressing anti-microbial transgenes (at moderate/high level) in the engineered plants is gradually increasing . Usuallyant cell . For exaant cell , a chimeponse 4)  and Glucse (GCB) . The chise (GCB)  has beenco lines . Likewis cassava . In this cassava .Brassica napus conferred resistance toward Rhizoctonia solani  when infected by the P4 strain of UMV4 totivirus  to generate a chimera ‘MUASCsV8CP’. The activity of this newly designed chimeric promoter (coupled to a GUS reporter) was evaluated in the transient (protoplast and agro-infiltration) and transgenic assays. Further, this recombinant promoter was employed for raising transgenic tobacco lines expressing Supplementary Table S1) adding EcoRI and HindIII sites at the 5′- and 3′- ends, respectively. The PCR amplified products were subsequently cloned in the EcoRI and HindIII sites of pUC119 sequencing vector and subsequently sequenced and analyzed to check their integrity. The fragments were finally sub-cloned into protoplast and plant expression vectors pUCPMAGUS and pKYLX71GUS  and three 3′- (CsVMVR1-CsVMVR3) end deletion fragments were PCR amplified  containing overhangs of EcoRI and HincII at the 5′- end and SmaI and HindIII at the 3′- end. The PCR amplified fragments were cloned into the corresponding sites of pUC119 following a previously published protocol  and the proximal domain of CsVMV8-Flt  promoter were PCR amplified using appropriate primers ] was fused with a 35-nucleotide untranslated region of AlMV RNA4  and apoplast targeting sequence (aTP) of Arabidopsis 2S2 protein gene (XhoI and SstI sites of the binary vector pKMUASCsV8CPGUS and pKCaMV35S2GUS (replacing GUS), to design the plasmids pKMUASCsV8CP-KP4His and CaMV35S2-KP4His. The pKMUASCsV8CP construct was also designed to serve as a vector control (VC).To generate the recombinant ein gene  at its 52 (cloned in pUCPMAGUS) were electroporated into viable tobacco protoplasts following standard protocols  were isolated and GUS activity analysis was performed according to standard protocols (2 generation) was performed according to a previously published protocol  were considered to be true transgenic lines. Independently re-generated transgenic lines showing proper segregation ratios (KanR:KanS::3:1) were selected and respective T2 generation transgenic plants were propagated in greenhouse conditions . The transgenic tobacco plants expressing KP4-His along with VC lines were also compared for their agronomical parameters such as plant height, leaf architecture, seed fertility and flowering time. Likewise, the GUS activities of the transgenic plants expressing pKYLXGUS, pKMUASCsV8CPGUS, pKMUAS35SCPGUS, pK35SGUS, and pK35S2GUS was performed following the standard protocol . Transgenic lines expressing native and chimeric promoter constructs along with KP4-His and VC plants were subjected to PCR amplification (of the genes mentioned above) following the standard protocol using Taq DNA Polymerase  having proofreading activity  overnight (16 h) at 37°C, electrophoresed on 0.8% agarose gel, blotted on nylon membrane  and finally probed with PCR amplified αP32-labeled GUS and KP4-His probes using specific primers (Supplementary Table S1). The entire procedure of Southern Blotting was performed following a previously described protocol  method  and cDNA synthesis was performed. After that, Real-time PCR analysis was performed using Premix Ex TaqTM II  employing the Opticon-2Real-time PCR . The transcript levels for individual constructs were calculated by the 2) method  and expring gene .2 generation) of KP4L2#2, KP4L2#3, KP4L2#4, KP4L2#7, KP4L2#11, and VC were extracted and crushed in liquid nitrogen to make a fine powder. Next, the total soluble protein (TSP) was extracted from the crushed samples using an extraction buffer containing 1X PBS with 0.05% β-mercaptoethanol and plant protease inhibitor cocktail (Sigma- P9599-5mL), pH 7.0. An aliquot of 10 μg of total soluble protein obtained from transgenic lines was resolved on 15% SDS-PAGE for Western blot analysis. For detection of the recombinant proteins, primary antibody specific to the hexa- (6x) His-tag [ and Rabbit polyclonal His-probe (SC-803)] was used following the standard protocol (The uppermost fully expanded leaves of 6-week-old greenhouse grown plants (Tprotocol .2) using Ni-NTA agarose (Qiagen) according to the manufacturer’s instructions with slight modifications as described earlier  buffer for 24 h at 4°C and adjusted to 40% ammonium sulfate saturation. After centrifugation at 30,000 g for 40 min, the enriched fraction obtained as pellet was solubilized, dialyzed (overnight) and filtered through a 0.45 μM membrane filter, and loaded on to a Ni-NTA agarose column. Finally, the column was washed with wash buffer . Ni-NTA agarose-bound KP4 was eluted with elution buffer . The eluted fractions were dialyzed extensively in 1x PBS and concentrated with a centrifugal filter device (Amicon 3 kDa cut-off).KP4 was purified from 21-days-old transgenic KP4-His seedling (T earlier . Briefly2) expressing KP4 was performed following the infiltration- centrifugation technique  and P. exigua var. exigua (MTCC No. 2321) cultures procured from MTCC  were grown on Potato Carrot Agar (PCA) medium (HiMedia- M696-500G) for 3 and 5 days, respectively. Fine wells were cut into the agar with the help of a cork borer (diameter = 0.5 cm) and 100 μl of the enriched test plant material prepared from both transgenic and VC lines were added to each well on either side of the fungal growth. Two other wells containing 100 μl each of sterile water and PBS were used as controls. The plates were then incubated at 25°C under constant light for A. alternata and, 12:12 h light:dark cycle for P. exigua var. exigua. The plates were photographed when clear zones of inhibition started to appear around the point of application of test samples.For agar-based killing zone assay, we followed a previously published protocol with slight modifications . The pla2, we cloned the KP4-His downstream of the above promoters to generate pKMUASCsV8CP-KP4His and pKCaMV35S2-KP4His constructs. The recombinant constructs were then agro-infiltrated in healthy tobacco leaves. After 3–4 days of incubation, the infiltrated leaf segments were excised and total protein was isolated as described above. Equal concentrations of protein from both the constructs were used for the agar-based killing zone assays against A. alternata and P. exigua var. exigua.To compare the protection efficacies of MUASCsV8CP and CaMV35SA. alternata and P. exigua var. exigua) were performed according to a standard protocol with minor modifications  and agar plugs containing A. alternata and P. exigua var. exigua were placed on the adaxial surface of the leaves. In case of P. exigua var. exigua assay, the leaves were wounded with the help of a sterile needle at multiple points (6–7). Alongside, a mock leaf was kept to evaluate the effects of wounding inflicted by the needle. The petri-plates were sealed and kept in dark at 28°C. The leaves were photographed on the 14th and 18th day post-inoculation (dpi) of A. alternata and P. exigua var. exigua, respectively. The lesion area percentage was calculated with the help of millimeter graph paper method , light intensity: 220 μmol/m2/s, temperature: 25 ± 2°C and humidity: 90–95%] were kept extremely humid and the symptoms were recorded/photographed 20 dpi. The lesion area percentage was recorded as described above.The whole plant assays were performed following a previously published protocol. Healthy leaves of 8-weeks-old transgenic KP4L2#2, KP4L2#3, and VC plants were sprayed with conidial suspension of . exigua  using a P-value of <0.05 was considered as significant.All experiment procedures were carried out in at least three independent replicates and the statistical analyses was performed using one-way analysis of variance (ANOVA) where a Figure 1A) were evaluated in tobacco protoplasts (cv. Xanthi Brad). Figure 1B depicts the average GUS activities obtained for each of the deletion constructs along with the CaMV35S promoter and an empty vector (EV) control with respective standard deviations (SDs). The data obtained clearly suggests that the relative GUS activity of the CsVMV8 (-215 to +166) was almost equivalent (∼1.1 times) to that obtained for the CaMV35S promoter; while the CsVMV7 (-256 to +166) ranked second. Furthermore, the TATA-less fragments CsVMVR2 (-565 to +16) and CsVMVR3 (-565 to -34) showed diminished GUS activities.The transient activities of each of the sixteen 5′ and 3′-end deletion constructs coupled to GUS reporter (pUCPMAGUS-CsVMV1 to pUCPMAGUS-CsVMVR3)  CsVMV1L4, CsVMV7L7, CsVMV8L6, CsVMV8L13, CaMV35SL9, and CaMV35S2L11. Finally, from the Southern-blot analysis, those transgenic plants having single copy insertion of the transgene (uidA) and exhibiting appropriate segregation ratio (3:1), namely CsVMV1L4, CsVMV7L7, CsVMV8L13, and CaMV35SL9 were chosen for further analyses .Finally, we raised transgenic tobacco plants by Agrobacterium mediated leaf disk transformation protocol as described in Methods. We successfully raised 7–10 independent transgenic lines expressing 2 generation). On analysis of the results, we observed that the CsVMV8 showed ∼1.1 times stronger activity than that of the CaMV35S promoter . Additionally, we performed qRT-PCR analysis of the transgenic plants as described in section “Materials and Methods.” The relative accumulation levels of uidA-mRNA level in transgenic plants (T2 generation) harboring the respective promoter constructs  confirmed ∼1.1-fold more expression of uidA under the control of CsVMV8 promoter in comparison to the CaMV35S promoter . Finally, we obtained a fairly strong intensity of X-Gluc staining of 21 days-old transgenic seedlings expressing the CsVMV8 promoter , in comparison to that obtained under other constructs.We evaluated the stable GUS activities of 3-week old transgenic tobacco seedlings  and in whole plants of tobacco, petunia, and tomato , we detected that the GUS activity of MUASCsV8CP was ∼2.4 times stronger as compared to the CaMV35S promoter.2-GUS (a modified version of CaMV35S with double enhancer domain) (uidA transgene in above plant lines (T1 generation). The appearance of distinct southern-positive bands in MUASCsV8CPL4 and MUASCsV8CPL6 indicate integration of the transgene construct as a single copy insertion in the T1 progeny plant genome . The progenies (T2 generation) of line MUASCsV8CPL6 exhibited expected segregation ratio and was chosen for further analyses.We raised transgenic plants harboring the MUASCsV8CP-GUS and CaMV35S domain)  construc2 transgenic seedlings having the CaMV35S, CaMV35S2, MUASCsV8CP, and MUAS35SCP promoters. The data obtained clearly showed that the chimeric promoter MUASCsV8CP, reported in this study has ∼2.4 and ∼1.1 times enhanced GUS activity as compared to CaMV35S and CaMV35S2 promoters, respectively. The data also indicates toward the equivalence in activities of MUASCsV8CP and MUAS35SCP promoters .We measured the GUS activities of the respective constructs using 21 days-old TX-Gluc staining of the transgenic seedlings expressing GUS under the control of CaMV35S, CaMV35S2, MUASCsV8CP, and MUAS35SCP promoters. The fold change as shown in Figure 4C indicates that the uidA-mRNA accumulation in MUASCsV8CP was nearly 1.8 times and 1.1 times as compared to CaMV35S and CaMV35S2 promoters, respectively, while MUASCsV8CP and MUAS35SCP showed the previous trend of being equivalent. Finally, the histochemical staining also suggested strongest intensity using MUASCsV8CP and MUAS35SCP chimeric promoters .Additionally, we performed the Real-time PCR analysis and histochemical 1 generation transgenic tobacco plants (KanR) expressing totiviral KP4 driven by MUASCsV8CP. Figure 5A represents the schematic map of both KP4-His and VC constructs. We performed segregation analysis of T1 generation transgenic plants as described in section “Materials and Methods.” Out of eighteen transgenic lines, the progenies (T1 generation) of eight independent lines showed proper segregation ratio (KanR: KanS::3:1) and found to be Kanamycin (300 mg/L) resistant on half strength Murashige Skoog (MS) medium (Supplementary Table S2). Seeds from these selected plants (T1 generation) showing proper segregation ratios were propagated to get homozygous T2 transgenic plants.Using Agrobacterium-mediated gene transformation method we raised 18 independent T) medium  (SupplemKP4 (537 bp), nptII (822 bp), and rbcSE9 (646 bp) (Supplementary Table S1). We confirmed the integration of different components of KP4 expression construct in T2 progenies of transgenic plant lines KP4L2#2, KP4L2#3, KP4L2#4, KP4L2#7, and KP4L2#11 whereas no amplification for KP4 was observed in the VC plant line . We analyzed the sequence integrity of the amplicons and did not detect any mutations in the nucleotide sequences of KP4, nptII, and rbcSE9.The integration of transgenes was confirmed by PCR amplification using primers specific to KP4-His transgenic lines and a VC plant (T1 generation progenies). Southern analysis confirmed the single copy insertion of KP4-His transgene construct in KP4L1, KP4L2, KP4L5, KP4L7, KP4L12, KP4L13, and KP4L15 transgenic tobacco plants . KP4L4 however, showed two copies of the transgene. Further, the transcript analysis of KP4 in KP4L2 transgenic lines  was performed by Real-time PCR. The result obtained from independent transgenic lines KP4L2#2, KP4L2#3, KP4L2#4, KP4L2#7, and KP4L2#11 clearly showed the accumulation of KP4 in respective transgenic lines in the following order: KP4L2#2 > KP4L2#3 > KP4L2#7 > KP4L2#11 > KP4L2#4 as shown in Figure 5D.Next, Southern-blot analysis was performed with gDNA isolated from different 2 generation). After isolation and quantification of TSP, we performed western blot analysis using anti-His polyclonal  primary antibody as described in section “Materials and Methods.” The KP4-His expressed in transgenic tobacco plants representing 2S2aTP-KP4-His was seen as a prominent band, the molecular mass being ∼18 kDa along with a few faint non-specific bands. To avoid the appearance of any contaminating bands, we performed western blot with anti-His monoclonal primary antibody . In this case, a discrete single band of ∼18 kDa indicating the presence of 2S2aTP-KP4-His in TSP was confirmed.In order to detect the KP4-His protein in transgenic plants, we isolated and concentrated TSP from individual transgenic seedlings  facilitated the purification of recombinant KP4 using Ni-NTA resin. The SDS-PAGE analysis clearly showed a band of ∼18 kDa after 40% ammonium sulfate enrichment coupled to Ni-NTA affinity chromatography process . We also observed a prominent contaminating band of ∼55 kDa which we assume might be of the RuBiSCO large subunit.To further detect the presence of KP4 in transgenic plants we performed nickel-nitrilotriacetic acid (Ni-NTA) affinity purification using TSP obtained from 21 days-old KP4L2#2 TArabidopsis 2S2 protein gene facilitated the accumulation of KP4 in the IF. The 40% ammonium sulfate enrichment and Ni-NTA affinity purification module was adopted for purifying KP4 from the IF of transgenic plants. Appearance of distinct bands of ∼18 kDa in the eluted fraction  indicated that the KP4 is properly targeted to the plant apoplast or intercellular space. In this case also we observed the band of ∼55 kDa as a contaminant.In order to confirm the accumulation of KP4 in the IF of transgenic plants, we isolated and partially purified the intercellular fluid  from the transgenic line KP4L2#2. The presence of 2S2aTP from A. alternata and P. exigua var. exigua using the ‘plate-diffusion’ assay  and Figures F (P. exigua var. exigua). It was evident that the KP4 activity represents a clear zone of inhibition around the point of application. This inhibition might be due to the blockage in calcium uptake by the fungal cells causing precocious hyphal disintegration.As KP4 blocks voltage-gated calcium channels of fungi, we sought to check the efficacy of plant-derived KP4 against two deuteromycetous fungal phyto-pathogens: n’ assay . Enriche2 promoters in the frame of fungal inhibition against A. alternata and P. exigua var. exigua. For this assay, we used transiently expressed KP4 from tobacco leaves infiltrated with pKMUASCsV8CP-KP4-His and pKCaMV35S2-KP4-His constructs . Although we observed a slightly enhanced inhibition in case of pKMUASCsV8CP-KP4-His, we assume equivalence in activities of both promoter constructs.Using KP4 as the gene of interest, we checked the efficacies of the MUASCsV8CP and CaMV35SAlternaria and hemi-biotrophic pycnidial pathogen Phoma causes foliar diseases in a wide variety of vegetables and annual plants , we checked for their virulence toward infecting tobacco plants. We found that both the pathogens could successfully infect mature tobacco leaves and develop visible symptoms (data not shown).Deuteromycetous necrotrophic fungal pathogen l plants . A. alteA. alternata, yellowish halos began to appear 7–8 days dpi while in P. exigua var. exigua, irregular brown spots appeared 10–11 dpi. The lesion area was measured 2 weeks post-inoculation for A. alternata  while 18 dpi for P. exigua var. exigua . The results revealed a high degree of protection in transgenic plants expressing KP4 as compared to the VC, as supported by the observation that the leaves of transgenic plants were found to be unaffected or developed smaller lesions than the VC plants.The disease resistance assays for both the pathogens were carried out with detached leaves kept on wet paper towels on petri dishes as described in section “Materials and Methods.” The detached-leaf technique is a routinely used method to determine the disease resistance of a particular transgenic plant against necrotrophic pathogens . In caseFigures 8C, 9C). These results suggested an enhanced anti-fungal activity of KP4 against these foliar fungal pathogens, which can be considered as significant.Following detached leaf assay, the lesion area percentage was determined using ‘Millimeter graph paper method’ as described in section “Materials and Methods.” We found, lesser lesion area in case of KP4 transgenic leaves as compared to the control plants . On an average, we observed that the VC plants showed 16.4% lesion area, while KP4L2#2 and KP4L2#3 showed 5.5 and 5.9% lesion areas, respectively . Likewise, we observed higher degree of lesions caused by P. exigua var. exigua in VC plants as compared to the KP4 transgenic lines KP4L2#2 and KP4L2#3 . We observed 12.4% lesion area  in VC plants, in comparison to 6.7 and 5.7% lesion areas in KP4L2#2 and KP4L2#3 transgenic lines, respectively .As described in section “Materials and Methods,” three independent experiments were carried out with whole plants of transgenic and VC lines. In all sets of experiments, the VC leaves developed distinct lesions much earlier than the A. alternata and P. exigua var. exigua, we found significantly lesser leaf-area exhibiting symptoms in case of KP4 transgenic leaves as compared to the VC plants. Although, some of the leaves showed inconsistent results, the percent lesion area clearly suggested inhibition and/or delayed lesion development in transgenic plants expressing KP4. The inhibition found in detached-leaf and whole plant assays further proved the anti-fungal efficacy of KP4 against both A. alternata and P. exigua var. exigua.Consistent with our results of detached-leaf assays for glucanase gene from alfalfa  with higher activity. It showed different levels of constitutive expression in different plant parts as follows: leaf > stem > root . Owing to the above features, it may appear as an important tool for plant modification. It can be efficiently used in combination with other caulimoviral promoters like CaMV35S and/or CaMV35S2 for co-expressing multiple genes (gene pyramiding) in a plant cell to avoid unwanted genetic recombination. Furthermore, the MUASCsV8CP can stand as a substitute for CaMV35S2 promoter in plant biotechnology-based applications as its activity is nearly comparable to that of the latter. Our in vitro agar-based well diffusion assays revealed that the recombinant KP4 expressed under both CaMV35S2 and MUASCsV8CP promoters showed almost equal zone of inhibitions against A. alternata and P. exigua var. exigua, suggesting that the newly designed promoter can also be capable of imparting protection against phytopathogens.Targeted gene expression in a transgenic platform lies under the control of the key genetic regulator ‘promoter’ which determines its spatio-temporal regulation. Designing of synthetic promoters can be of immense importance in plant modification through genetic engineering that can withstand biotic stress while also ensuring maximum productivity in the field of translational biology . In thisKP4 was chosen as the gene of interest based upon the inter-strain hindrance found in double-stranded DNA totiviruses. Having structural similarity to the scorpion toxin AaHII, it was proposed and eventually proved that KP4 prevents uptake of calcium by fungal cells. As a result, it prevents hyphal tip growth, budding and viability of the fungi. KP4 has no sequence similarity to any other member of the ‘killer protein’ gene family and is extremely basic in nature with a pI of 9.0. The KP4 is known to be composed of five-stranded antiparallel β-sheet and two antiparallel α-helices that are stabilized by five disulfide bonds. It has been reported that KP4 has no apparent effect on the viability or subcellular structures of human, plant or insect cell lines as well as found to be harmless to soil inhabitants. Moreover, KP4 degrades in the stomach fluid and has no sequence resemblance with any known allergens . However, this delay was not as evident as observed by 1 progenies) indicated the single copy insertion of the transgene construct. We chose KP4L2 for our further studies as it exhibited good growth and maximum number of homozygous lines. Molecular analysis of the transgenic plants expressing KP4 confirmed the integration of KP4 in T2 progenies of KP4L2 lines viz. KP4L2#2, KP4L2#3, KP4L2#4, KP4L2#7, and KP4L2#11. Subsequently, Real-time PCR analysis of the above transgenic plants indicated toward the maximum transcript accumulation in KP4L2#2.A total of eight transgenic plant lines harboring MUASCsV8CP-KP4His  showed normal agronomical parameters with proper segregation ratio but slight delay in root growth .Plants monitor the apoplastic fluid, secrete hydrolytic enzymes and anti-microbial metabolites in response to fungal colonization which re-models the apoplast to become the first battleground for any plant-fungal interaction . Hence, ein gene . Also, oA. alternata and P. exigua var. exigua in the in vitro agar plate diffusion assays. This report is in accordance with the earlier published results of Fusarium graminearum, the causal agent of devastating head blight of small grain cereals , which causes vascular wilt in a wide range of plant species  which may be due to its ability to form long-lasting resistant melanised clumps called microsclerotia in soil/media  was found to be the highest expressing fragment; secondly, hybridization of the CsVMV8 fragment with UAS of MMV (-297 to -38) to develop an inter-molecularly shuffled recombinant promoter “MUASCsV8CP” and finally, expression of the totiviral KP4 under this chimeric promoter to develop transgenic tobacco resistant against two foliar pathogenic fungi – IM and ND conceptualized the experiments. DD, AS, and ND designed the experiments and critically analyzed the results and wrote the paper. DD performed all the experiments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Diexf knockout mice. We demonstrate that, similar to Mdm2 and Mdm4, homozygous deletion of Diexf is embryonic lethal. However, unlike in Mdm2 and Mdm4 mice, loss of p53 does not rescue this phenotype. Moreover, Diexf heterozygous animals are not sensitive to sub-lethal ionizing radiation. Thus, we conclude that Diexf is an essential developmental gene in the mouse, but is not a significant regulator of the p53 pathway during development or in response to ionizing radiation.Increased levels of inhibitors of the p53 tumor suppressor such as Mdm2 and Mdm4 drive tumor development and thus serve as targets for therapeutic intervention. Recently, digestive organ expansion factor (Diexf) has been identified as a novel inhibitor of p53 in zebrafish. Here, we address the potential role of Diexf as a regulator of the p53 pathway in mammals by generating  At the same time, p53 transcriptional activity is tightly regulated in normal cells to prevent inappropriate activation of downstream anti-proliferative or cell death programs . The twop53 , 7. In herations . Howeverentified .Diexf), was identified as a novel inhibitor of p53 in zebrafish [DIEXF is an evolutionarily conserved gene that was first identified in zebrafish and shown to be essential for expansion of digestive organs [Recently, digestive organ expansion factor  had TP53 alterations and 25% (n = 513) had Diexf amplification, but only 6% (n = 134) had both  was present between DIEXF amplification and TP53 alterations. Significant mutual exclusivity was also observed in other breast cancer datasets in Figure DIEXF is located on chromosome 1q in close proximity (5.3Mbp) to the MDM4 locus. It is thus possible that DIEXF amplification is a consequence of co-amplification with MDM4. When we analyzed the same breast cancer datasets, we observed that amplification of DIEXF and MDM4 significantly overlapped  in mice. Exon 1 is 229 base pairs and contains the translational start site. We selected two sequences in Exon 1 downstream of the start codon to target with specific sgRNAs and the spCas9 endonuclease  resulting in a premature stop signal after 85 amino acids (23 endogenous and 62 novel amino acids). The second allele Diexf13ins had a 13 base pair insertion between bases 200–201 in exon 1 resulting in premature stop after 28 amino acids (26 endogenous and 2 novel amino acids).To directly evaluate the function of Diexf and its potential role in regulation of the p53 pathway e Figure  respectisis tool . When bod Figure . Of 16 acrispr.mit.edu analysis tool . Failure to generate any viable homozygous knock-out animals clearly demonstrates an essential role of Diexf during embryonic development in mice.The two selected lines of Diexf+/∆26 heterozygous crosses at various developmental time points. We screened 13 embryos from 2 females at E14.5 and did not find any homozygous mutants (Table n = 9) or heterozygous mutants (n = 15). The finding of empty decidua demonstrated that mutant embryos implanted but died soon thereafter and were likely resorbed by E7.5 [Diexf null embryos are peri-implantation lethal at E4.5-E5.5 stages. To further evaluate the viability of mutant embryos at pre-implantation stage, we flushed out blastocysts from heterozygous crosses, observed the morphology under microscope, and genotyped them. We did not observe obvious defects in dozens of blastocysts examined, and genotyping of 8 random blastocysts confirmed presence of homozygous mutants  upon p53 loss in Diexf null embryos, we also screened embryos from Diexf+/∆26; p53+/- double heterozygous mice crossed with Diexf+/∆26; p53-/- animals at various time points. We failed to find any embryos with the Diexf∆26/∆26 genotype irrespective of p53 status at the time points examined, and empty decidua were observed at E7.5 and E9.5 as previously observed with the Diexf+/∆26 heterozygous crosses  in a p53-dependent manner [Diexf+/∆26 heterozygous animals (n = 14) along with Mdm2P2/P2 animals (n = 5) as a positive control [n = 12) as a negative control. Mice were irradiated with sub-lethal IR (one dose of 6 Gy) and monitored daily for four weeks. As expected, radio-sensitive Mdm2P2/P2 animals died within three weeks after irradiation  and A. thaliana (Nof1) are nucleolus-localized components of small subunit processome that regulate pre-rRNA processing [DIEXF amplification and TP53 mutation in human breast cancers. This relationship must, however, be considered in the context of the DIEXF genomic locus. DIEXF is in close proximity to MDM4 gene which encodes a potent p53-inhibitor that is frequently amplified in human cancers [DIEXF and TP53 is driven by amplification of MDM4. We cannot, however, exclude the possibility that overexpression of DIEXF contributes to p53 pathway attenuation in these cancers.Recent studies show that Diexf homologs in ocessing , 18, 28.ocessing , 17, 29. cancers , 31. GivDiexf knockout alleles in the mouse. The CRISPR/Cas9 system has revolutionized the way we generate animal models as it is efficient, fast, and less expensive than traditional techniques. One limitation of this system, however, is the difficulty in generation of knock-out alleles of genes that may be essential. If both alleles of an essential gene are disrupted, the embryos may not survive. Using only sgRNA1 and spCas9 endonuclease, we failed to generate any mice with disruption of Diexf gene. We could not determine whether the sgRNA failed to target the gene or if the sgRNA was so efficient that bi-allelic disruption of the gene in all targeted mice led to lethality. When both sgRNA1 and sgRNA2 were co-injected, only sgRNA2 generated mutations at its target site indicating that sgRNA1 failed. Moreover, even though both alleles were targeted by sgRNA2 in many animals, one or both alleles were always in-frame resulting in embryo survival. If most mutations had not been in-frame, more embryos would have died. We observed the same 9 bp deletion in 10/16 mice. The observation that non-homologous end joining (NHEJ) based repair can result in frequent recurrence of the same mutation in targeted alleles also presents a problem. If the recurring mutation is frame-shift, then more embryos would die. On the other hand, if the recurring mutation is in-frame, the possibility of getting a knock-out allele is diminished. Therefore, both lethality and sgRNA failure should be considered while targeting a possible essential gene. Another major concern of the CRISPR/Cas9 system is the modifications (insertions or deletions) at off-target sites. To our knowledge, there has been no report of high frequency targeting at non-targeted sites with 2 or more mismatches in mouse models. When we screened the top four and the top six predicted off-target sites for sgRNA1 and sgRNA2 respectively, we did not find any mutations.To directly evaluate the role of Diexf as a regulator of the p53 pathway in mammals, we used CRISPR/Cas9 technology to generate multiple independent Diexf is peri-implantation lethal, establishing Diexf as an essential developmental gene in the mouse. Our observation that Diexf-null mouse embryos are peri-implantation lethal is very different from the phenotype observed in zebrafish. In fish, the lethality occurs due to digestive organ failure, while in mice, the embryos died before any of the digestive organs develop. In addition, the abnormalities in mutant fish are p53-dependent, as the phenotype can be partially rescued by concomitant p53 loss. However, embryonic lethality in mice due to Diexf loss was not rescued or even delayed by loss of p53 demonstrating that the phenotype is not a consequence of inappropriate activation of p53. The role of p53 regulators such as Mdm4 seem to have evolved from fish to mammals as well. A recent study has shown that Mdm4 is dispensable in zebrafish, while it is essential for murine embryonic development [Using two independent null-alleles, we demonstrate that homozygous loss of elopment , 25. We Diexf heterozygous animals do not exhibit enhanced radio-sensitivity, further supporting the argument that Diexf is not a potent inhibitor of the p53 pathway in mice under these conditions. In human cancers, negative regulators of the TP53 pathway are frequently overexpressed, so it remains possible that the overexpression of DIEXF may impact the TP53 pathway and have pathological consequences. Moreover, it may inhibit TP53 function under certain conditions that we have not tested.Finally, we find no evidence of Diexf as a significant functional regulator of the p53 pathway in response to ionizing radiation. Heterozygous loss of well-established p53 regulators results in sensitivity to a sub-lethal dose of ionizing radiation . Diexf hDiexf as an essential developmental gene in the mouse, and suggests that the function(s) of Diexf are largely independent of a role as a negative regulator of p53. It has been clearly demonstrated that Def is a component of the ribosomal processome, and the Def-Capn3 pathway possibly regulates proteins other than p53 in the nucleolus. Two previous publications with Capn3 deletion in mice show that the Capn3-/- mice are viable and fertile, and their digestive organs are not affected [Diexf-null mice is independent of both p53 and Calpain3, and suggests that the role of Diexf has evolved to have very diverse functions. Moreover, as DIEXF amplifications occur in cancers, future studies to better understand its functions will be instructive and important.Combined, this work identifies affected , 33. Thecrispr.mit.edu tool. The two sites with highest scores were selected for targeting. Four and six possible off-target sites with a PAM sequence for sgRNA1 and sgRNA2 respectively were also selected for screening.The entire sequence of exon 1 (starting from 20 bases upstream of the start codon) was used to score all possible target sites using the in-vitro transcription kits use T7 polymerase which requires a G at the transcription start site. For sgRNA1, N20 does not start with a G, and thus G (in parenthesis below) was added to the sgRNA. The T7 promoter sequence was underlined, and sgRNA target sites are in bold. Four random bases (GAAA) were added upstream of T7 promoter sequence to provide anchorage for T7 polymerase binding. The following sequence is the Forward oligo for the template. Reverse complement of this sequence will be the reverse oligo.All TTAATACGACTCACTATA(G)ATGGGCAAACGCCGGAACCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTGAAATTAATACGACTCACTATAGCACCTCCGCGACTTCGGCGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTGAAAOligos were suspended in nuclease free water to a final concentration of 500 ng/µl. For each sgRNA, 5µg each of forward and reverse oligos (10 µl each) were mixed in 80µl nuclease-free water  and boiled for 5–10 minutes and then cooled at room temperature for 2 hrs to overnight. 200–400 ng of the prepared templates were used to synthesize sgRNAs using the MEGAshortscript Kit (Invitrogen AM1354) as per manufacturer’s protocol. sgRNAs were purified by acid phenol-chloroform extraction and ethanol precipitation as per manufacturer’s protocol (Invitrogen). sgRNAs were resuspended in 70 µl of RNase-free water and further purified by using Biospin P30 chromatography columns  as per manufacturer’s protocol to remove any remaining free nucleotides. The concentration and quality of sgRNAs were determined by using Bio analyzer. If the concentration measured by Nanodrop was significantly higher than Bio analyzer reading, it indicated free nucleotide contamination. Column purification was repeated if necessary.A final injection solution containing 10 ng/µl Cas9 mRNA  and 7.5 ng/µl of each sgRNA was prepared in Tris-EDTA buffer . The final solution was injected into the pronucleus of 200–250 zygotes. The zygotes were then implanted into pseudo-pregnant mice . All injections and implantations were done in the MD Anderson Genetically Engineered Mouse Facility.Mice were maintained in 100% C57BL/6 background. All mouse studies were conducted in compliance with an Institutional Animal Care and Use Committee protocol. Live mice were weaned at the age of three weeks, and ear biopsies were collected. The tissues were digested in Lysis buffer  at 55°C overnight, and heated at 95°C for 15 minutes to denature Proteinase K. 2 µL of the lysed tissue extract was used for PCR reaction to amplify 1 Kb region of the targeted site. The PCR product was gel purified and sanger-sequenced to identify any indels at the target site.Diexf∆26 Genotyping primer Fwd: CGTTTCCGCTATGGGCAAACG; Diexf∆26 Genotyping primer Rev: CTCAACTCGGCCGGAACCAG.Screening primer Fwd: CGCATGCGTAGACACGCCTATG; Screening primer Rev: GCACAAGGGCAGAGATGATCAG; crispr.mit.edu site for both sgRNAs. The following criteria were used to select the sites for screening:A list of all possible off-target sites was obtained from 1. All sites with less than 3 mismatches were selected; 2. Sites with 3 or more mismatches should be followed by PAM sequence; 3. Intra-genic sites were given preference over inter-genic sites; 4. Sites with higher score were selected first; 5. For sites with the same score, one with fewest mismatches was selected; 6. For sites with the same score and the same number of mismatches, the average of the mismatch positions were considered (one with lowest average was selected).Once the sites were selected, the 23 base sequence for each site was run on BLAT tool at UCSC Genome Browser website, and the genomic sequence was obtained. Primers were designed about 500 bases upstream and 500 bases downstream of the selected sites, and the region was PCR amplified and sanger-sequenced.sgRNA1 OTS1 Fwd: CCTCCACCCCGCTCTAATTTC; sgRNA1 OTS1 Rev: CTGCCCTTCTCCTCTGTGGATC; sgRNA1 OTS2 Fwd: GGGAAGGAAGCTCAGGGGTTAG; sgRNA1 OTS2 Rev: CCACCTTGGAATTCCGTTCTTTC; sgRNA1 OTS3 Fwd: GGAGGCAGTGAAAGATAAAG; sgRNA1 OTS3 Rev: CGGATCACTCAGTTTGAATC; sgRNA1 OTS4 Fwd: CGGGTTGCTTTGGAAAAAATACAC; sgRNA1 OTS4 Rev: CGGGCTGACCGTATTGAGGGAATC; sgRNA2 OTS1 Fwd: GGCTGTGGTAGGTGATTAC; sgRNA2 OTS1 Rev: GGTCACCAGCTAAGGAATG; sgRNA2 OTS2 Fwd: GACCCGGGTAAGAAGAAAAAG; sgRNA2 OTS2 Rev: CGGCGAAACAGACTGTTTC; sgRNA2 OTS3 Fwd: CACACAGGATGTCACATTCC; sgRNA2 OTS3 Rev: GCGAGCTGCCCTTTTAAG; sgRNA2 OTS4 Fwd: GCCATGCAACCTCCTAAG; sgRNA2 OTS4 Rev: CTCCAGGATCTTGCTTTTGG; sgRNA2 OTS5 Fwd: CTGGGACTAGTTTCTGGACTC; sgRNA2 OTS5 Rev: GCACACCCTGTATCTAACATTG; sgRNA2 OTS6 Fwd: GGGGCAGAGACAATCATG; sgRNA2 OTS6 Rev: GGCTGAGAATGGCTCAAG.Heterozygous animals were mated with each other, and plugs were examined every morning. Females positive for plug (E0.5) were euthanized 7 days (E7.5) or 9 days (E9.5) from that date. Uteri were dissected, and each decidua was separated and dissected as previously described . EmbryosAs described previously , 8–12 we5–9 week old mice were exposed to one dose of 6 Gy IR in a cesium-137 irradiator and monitored daily for four weeks. Moribund animals were sacrificed.Protein lysates were prepared by lysing tissues in SDS buffer. Protein estimation was carried out with BCA . Seventy micrograms of lysate was resolved on 8% SDS-PAGE and immunoblotted with antibodies against Diexf  and Vinculin .t-tests and Kaplan-Meier survival analyses were performed using Prism 5 software (GraphPad Software). P-values < 0.05 were considered statistically significant.Student’s


Escherichia coli CNT family member NupC resembles hCNT1 in permeant selectivity but is H+-coupled. Using heterologous expression in Xenopus oocytes and the engineered cysteine-less hCNT3 protein hCNT3(C−), substituted cysteine accessibility method analysis with the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate was performed on the transport domain  7, and TM8), as well as TM9 of the scaffold domain of the protein. This systematic scan of the entire C-terminal half of hCNT3(C−) together with parallel studies of the transport domain of wild-type hCNT1 and the corresponding TMs of cysteine-less NupC(C−) yielded results that validate the newly developed structural homology model of CNT membrane architecture for human CNTs, revealed extended conformationally mobile regions within transport-domain TMs, identified pore-lining residues of functional importance, and provided evidence of an emerging novel elevator-type mechanism of transporter function.The human SLC28 family of concentrative nucleoside transporter (CNT) proteins has three members: hCNT1, hCNT2, and hCNT3. Na Specialized nucleoside transporter proteins are required for passage of nucleosides and hydrophilic nucleoside analogs across biological membranes. Physiologic nucleosides serve as central salvage metabolites in nucleotide biosynthesis, and nucleoside analogs are used in chemotherapy of cancer and antiviral diseases , 2. Aden+-dependent nucleoside transporters found predominantly in intestinal and renal epithelial and other specialized cell types 5–ll types , 12. hCNhiometry –19. hCNThiometry , 19, 20.+-linked CNT from Vibrio cholerae (vcCNT) with bound uridine and sodium was determined at a resolution of 2.4 Å +-linked By analogy with the bacterial structure, hCNT3 topology can be subdivided into an outer or “scaffold” domain comprising TM4, TM5, IH1, TM6, and TM9, which surrounds an inner or “transport” domain that can itself be divided into two structural subdomains that are related by an internal 2-fold pseudo-symmetry. The first subdomain comprises IH2, HP1, TM7, and TM8, whereas the second subdomain comprises IH3, HP2, TM10, and TM11 . hCNT3 rEptatretus stouti, have revealed that the functional domains responsible for CNT nucleoside selectivity and cation coupling reside within the C-terminal HP1-TM11 half of the protein  . Employed originally in a yeast expression system for substituted cysteine accessibility method (SCAM) analysis of what is now recognized as the second or C terminus transport domain using methanethiosulfonate reagents , we haven system  and have (PCMBS) , 29. Mea (PCMBS) , 31.554, Tyr558, and Cys561) that exhibit H+-activated inhibition by PCMBS, with uridine protection evident for Tyr558 and Cys561 (519 in the HP2 region of hCNT3 and the corresponding residue in hCNT1 (Glu498) were also identified as having key roles in permeant and cation coupling/translocation (XKX3NEFVA(Y/M/F) , our results validate the newly predicted structural model of CNT membrane architecture, reveal extended conformationally mobile regions at the tip of HP1 and within TM7, identify further pore-lining residues of functional importance, and support an emerging novel elevator-type mechanism of transporter function.To complete investigation of the transport domain of hCNT3, the present study reports a comprehensive PCMBS SCAM analysis of the corresponding N-terminal transport subdomain of this transporter , as well as the long linker TM9 region of the protein. Together with parallel studies of HP1 and TM7 of NaE. coli NupC (NupC(C−)).All 14 endogenous cysteine residues of hCNT3 were replaced with serine to produce hCNT3(C−), a cysteine-less hCNT3 construct , 27. hCN+ and H+ electrochemical gradients  and Na+-free, acidified medium . The Na+-containing medium was buffered at a pH of 8.5 to avoid the small but significant H+ activation of hCNT3 shown previously to occur at pH 7.5  by oocytes producing hCNT3(C−) varied in different experiments between 2 and 4 pmol/oocyte·min−1; the corresponding uptake of uridine by water-injected oocytes was <0.02 pmol/oocyte·min−1 (data not shown).hCNT3 transports nucleosides using both Naradients , 20. Thet pH 7.5 , 20. In t pH 7.5 . Initial−1 were excluded from further analysis . In both cases, the mutation to cysteine resulted in a protein with low functional activity in both Na+-containing, H+-reduced and Na+-free, acidified media . The two residues were Glu343 in HP1b and Tyr379 in TM7b. Cell-surface labeling with sulfo-NHS-LC-biotin and immobilized streptavidin resin were used to distinguish cell-surface proteins from those associated with total (plasma + intracellular) membranes. Both mutants, which had electrophoretic mobilities similar to that of hCNT3(C−), were present at cell surfaces in greatly reduced amounts so that no conclusions could be reached regarding their transport activities (data not shown).Two mutants that exhibited uridine uptake values of <0.1 pmol/oocyte·minsupplemental Table S1 additionally presents uridine transport activity of each construct calculated as the flux ratio of Na+-mediated to H+-mediated uptake (Na+:H+). The corresponding Na+:H+ ratios of uridine uptake (10 μm) for wild-type hCNT3 and cysteine-less hCNT3(C−) were ∼1.7 and 1.0, respectively , and were consistent with results of previous studies  are highlighted in the hCNT3 topology schematic shown in To facilitate comparisons between the remaining 170 mutants,  studies , 27, 28.+:H+ uptake ratios similar to those of either hCNT3 or hCNT3(C−). In contrast, there were eight mutants in HP1 that gave Na+:H+ ratios of >2.5 (S330C(C−), G335C(C−), N336C(C−), V339C(C−), G340C(C−), Q341C(C−), S344C(C−), and P345C(C−) with values of 2.7, 3.5, 6.5, 2.9, 10.2, 4.6, 9.4, and 9.2, respectively), and two mutants, S334C(C−) and I337C(C−), that exhibited low Na+:H+ ratios of 0.3 and 0.4, respectively.Most constructs exhibited Na+:H+ uptake ratio of 4.5, and within TM7, mutants S369C(C−), T370C(C−), and V375C(C−) had values of 2.9, 0.2, and 8.0, respectively. One mutant in TM8 had a Na+:H+ ratio of >2.5 (L389C(C−), with a value of 5.9, and one mutant each in TMs 8 and 9, M395C(C−) and L461C(C−), exhibited low Na+:H+ ratios of 0.38 and 0.36, respectively. All mutants in the large loop linking TMs 8 and 9 resembled hCNT3 and hCNT3(C−).One mutant in the loop between HP1 and TM7 and a cluster of three mutants in TM7 also exhibited altered cation-coupling properties: loop mutant L352C(C−) gave a Nai.e. in Na+-free, acidified medium), there being no PCMBS inhibition in either Na+-containing, H+-reduced or Na+-free, H+-reduced medium  and in Na+-free, acidified medium . After 10-min exposures to 200 μm PCMBS, uptake of 10 μm radiolabeled uridine was assayed in medium of the same composition. Exposure to PCMBS was performed on ice to minimize its diffusion across oocyte plasma membranes  and hCNT3(C−) .Wild-type hCNT3 has previously been reported to be sensitive to inhibition by PCMBS under acidic conditions only  was PCMBS-inhibitable only in Na+-free, acidified medium. In TM7, nine adjacent mutants (I371C(C−), A372C(C−), G373C(C−), S374C(C−), V375C(C−), L376C(C−), G377C(C−), A378C(C−), and I380C(C−)) were PCMBS-inhibitable in both cation conditions, whereas the immediately adjacent residue, Thr370, was PCMBS-inhibitable upon conversion to cysteine only in Na+-free, acidified medium. One residue that localizes to the predicted extracellular loop following TM7  was sensitive to PCMBS inhibition upon conversion to cysteine in both Na+-containing, H+-reduced and Na+-free, acidified medium.In HP1, three adjacent residues, Gly+-containing, H+-reduced and Na+-reduced, acidified medium for four adjacent mutants in TM8 (L390C(C−), T391C(C−), A392C(C−), and S393C(C−)). In close proximity to this block of residues, three additional mutants (H388C(C−), V394C(C−), and M395C(C−)) were sensitive to PCMBS inhibition only in Na+-reduced, acidified medium. Within TM9, two mutants (I450C(C−) and L461C(C−)) were sensitive to PCMBS inhibition only in Na+-reduced, acidified medium. None of the residues in the TM8–9 loop, when converted to cysteine in hCNT3(C−), were inhibitable by PCMBS under either cation condition.PCMBS inhibition was also observed in both Nam) to protect against inhibition by PCMBS for hCNT3(C−) mutants that were inhibited in either or both cation conditions. Results for each individual mutant are presented in Subsequent experiments investigated the ability of extracellular uridine , which was also PCMBS-inhibitable under both cation conditions, was protected against PCMBS inhibition in Na+-containing, H+-reduced medium only.In HP1, three residue positions exhibited uridine protection from PCMBS inhibition. Q341C(C−) and T342C(C−), which were PCMBS-inhibitable in both Na+-free, acidified medium, exhibited full uridine protection against that inhibition, whereas A372C(C−), G373C(C−), and S374C(C−), which were PCMBS-sensitive in both Na+-containing, H+-reduced and Na+-free, acidified media, were fully protected under both cation conditions. In contrast, I371C(C−), which was inhibited by PCMBS under both cation conditions, was protected by uridine only in Na+-containing, H+-reduced medium.In TM7, five residue positions were uridine-protected. T370C(C−), which was PCMBS-sensitive only in Na+-reduced, acidified medium, showed full uridine protection, and T391C(C−) and S393C(C−), which were PCMBS-inhibited in both Na+-containing, H+-reduced and Na+-reduced, acidified media, were also fully uridine-protected under both cation conditions. In contrast, A392C(C−), which was also PCMBS-inhibited under both cation conditions, was protected by uridine only in Na+-containing, H+-reduced medium. None of the PCMBS-sensitive mutants in TM9 was protected by uridine.In TM8, five residue positions were protected against PCMBS inhibition by uridine. V394C(C−) and M395C(C−), which were PCMBS-inhibited only in Na+-free, acidified medium  and in either Na+-free, H+-reduced medium  or Na+-containing, acidified medium . Fluxes of 10 μm uridine were then determined under Na+-free, acidified conditions (Of the 24 hCNT3(C−) residue positions in the HP1-TM9 region that were PCMBS-sensitive, 17 were inhibited by PCMBS in both cation conditions, whereas 7 were inhibited by PCMBS only in Nad medium . To detenditions .+-containing, H+-reduced or Na+-free, acidified medium were also inhibited by PCMBS in Na+-free, H+-reduced or Na+-containing, acidified medium  in HP1; T370C(C−) in TM7; H388C(C−), V394C(C−), and M395C(C−) in TM8; and I450C(C−) and L461C(C−) in TM9) were unaffected by PCMBS under cation-reduced conditions  but strongly inhibited by PCMBS in Na+-containing, acidified medium as was previously seen for residues Ile554, Tyr558, and Cys561 in TM10 of hCNT3  (+-specific hCNT1 with those for Na+- and H+-dependent hCNT3 would identify features associated with the different cation-coupling characteristics of these transporters.Complementary to the above studies of hCNT3(C−), we also undertook parallel SCAM analyses of HP1 and TM7 of hCNT1 and of the two equivalent transmembrane regions (HP1 and TM4) of CNT3(C−) , 28. Thisupplemental Fig. S1. Single-cysteine mutants were scanned for functional activity in Na+-containing, H+-reduced medium  and tested for PCMBS inhibition and uridine protection under the same conditions . To determine whether access of PCMBS required Na+-induced conformational changes, inhibitor-sensitive mutants were rescreened for PCMBS inhibition under either Na+-containing, H+-reduced or Na+-free, acidified conditions . Media were buffered at pH 8.5 to enable direct comparison with data obtained for hCNT3(C−). Control experiments at pH 7.5 yielded essentially identical results (data not shown). Results from the experiments are summarized in the topology schematic of Fig. 6A, and compared with hCNT3(C) in the sequence alignment of −1, it was possible to test all 43 individual cysteine mutants for sensitivity to PCMBS inhibition.The 43 residues of hCNT1 spanning the region between and including HP1 and TM7 that were investigated in the present study are highlighted in 319, Gln320, Thr321, and Glu322. Of these, Gln320, Thr321, and Glu322 showed uridine protection (A). This clustering of adjacent PCMBS-inhibitable residues was similar to hCNT3(C−)   and 7 an350, Ala351, Gly352, Ser353, Leu354, and Leu355). Of these, mutants I350C, G352C, S353C, and L354C showed uridine protection. Again, the clustering of adjacent PCMBS-sensitive residues was similar to hCNT3(C−) and inconsistent with a conventional α-helix. Similarities between the two transporters also extended to the presence of a single PCMBS-sensitive residue in the putative external loop exiting TM7 ) . Residues that were sensitive to PCMBS inhibition in the presence of Na+ were also inhibited by PCMBS in its absence , indicating that access of PCMBS to hCNT1 inhibitor-sensitive residues did not require Na+.In TM7 of hCNT1, six adjacent residues were sensitive to PCMBS inhibition when mutated to cysteine  was replaced with either serine or alanine to produce cysteine-less NupC constructs C96S and C96A, respectively. Produced in Xenopus oocytes, C96S exhibited transport characteristics similar to those of wild-type NupC, but with lower functional activity, exhibiting a moderate ∼3-fold increase in uridine apparent mK, a kinetic deficit that was overcome by conversion of Cys96 instead to alanine (data not shown). NupC C96A (hereafter referred to as NupC(C−)), therefore, was used as template for the construction of single-cysteine mutants.Extending the analysis to Hsupplemental Fig. S2. Single-cysteine mutants were scanned for functional activity in Na+-free, acidified medium  and tested for PCMBS inhibition and uridine protection under the same conditions . To determine whether access of PCMBS required H+-induced conformational changes, inhibitor-sensitive mutants were rescreened for PCMBS inhibition under either Na+-free, acidified or Na+-free, H+-reduced conditions . Results from the experiments are summarized in the topology schematic of Fig. 6B and compared with hCNT3(C−) and hCNT1 in the sequence alignment of The 45 NupC(C−) residues spanning the region between and including HP1 and TM4 of NupC (corresponding to hCNT1/3 HP1 and TM7) that were investigated are highlighted in −1 and were excluded from further analysis . Five of these were in HP1 (E135C(C−), F137C(C−), G146C(C−), F151C(C−), and I152C(C−)), and 7 were in TM4 (M166C(C−), T172C(C−); S175C(C−), T176C(C−), M179C(C−), G183C(C−), and M186C(C)). Mutagenesis of NupC(C−) therefore resulted in a higher percentage of non-functional mutants when compared with mutagenesis of hCNT3(C−) or hCNT1. This may, in part, reflect production in a eukaryote heterologous expression system and the attendant lower overall transport activity exhibited by the bacterial transporter. However, as shown in the sequence alignment of + coupling is generally more sensitive to mutation than Na+-coupling, and because NupC is exclusively H+-dependent, this might also be a contributing factor to the larger number of non-functional NupC mutants.Of the 43 NupC(C−) mutants examined, 12 exhibited uridine uptake values of <0.05 pmol/oocyte·10 min142, and this inhibition was not uridine-protectable. This residue is not part of the cluster of adjacent residues found to be PCMBS-sensitive in hCNT3(C−) and hCNT1. It is, however, centrally positioned in the TM and precedes by one position a residue in hCNT3(C−) (Ile337) that was sensitive to PCMBS inhibition under Na+-reduced, acidified conditions.In HP1 of NupC(C−), PCMBS inhibition was evident only for residue position SerIn TM4 of NupC(C−), two pairs of adjacent mutants, S180C(C−)/I181C(C−) and A184C(C−)/Y185C(C−), were inhibited by PCMBS. Only I181C(C−) was protected by uridine. The corresponding residues in hCNT3(C) and hCNT1 are part of the cluster of adjacent residues that were also sensitive to PCMBS inhibition.+ were also inhibited by PCMBS in its absence . Access of PCMBS to NupC inhibitor-sensitive residues did not therefore require H+. One mutant, Y185C(C−), was more sensitive to PCMBS inhibition under acidified conditions but moderately inhibited by PCMBS in H+-reduced medium .Residues that were sensitive to PCMBS inhibition in the presence of Hm). Of the eight hCNT3(C−) cysteine mutants tested, five showed significantly different nucleoside uptake profiles compared with those of wild-type hCNT3 and hCNT3(C−).HP1 and TM7 of hCNT1/2 contain residues that determine the nucleoside preferences of the two transporters , 36. A f+-containing, H+-reduced medium (A and B). Both also exhibited the characteristic narrowing of nucleoside preference under Na+-free, acidified conditions  exhibited broad transport preference for both purine and pyrimidine nucleosides in Nad medium , 27 .+-containing, H+-reduced medium, whereas uptake was greatly reduced, although measurable, for all nucleosides tested in Na+-free, acidified medium (C). Similar to hCNT3 and hCNT3(C−), V375C(C−) exhibited broadly selective uptake of both purine and pyrimidine nucleosides in Na+-containing, H+-reduced medium, but only very low uptake of uridine, thymidine, adenosine and inosine, and no detectable transport of cytidine and guanosine in Na+-free, acidified medium (G).Three of the mutants with altered permeant selectivities were located within HP1 (G340C(C−), Q341C(C−), and T342C(C−)), and two were located in TM7 (S374C(C−) and V375C(C−)). Of these, G340C(C−) and V375C(C−) had the greatest differences compared with hCNT3 and hCNT3(C−). Relative to uridine, G340C(C−) exhibited reduced uptake of thymidine, adenosine, inosine, and guanosine in Nad medium C. Similad medium G.340 in HP1, mutants Q341C(C−) and T342C(C−) showed (i) reduced uptake of cytidine and thymidine (Q341C(C−)) or thymidine and inosine (T342C(C−)) in Na+-containing, H+-reduced medium and (ii) reduced uptake of uridine, cytidine, thymidine, and adenosine (Q341C(C−)) or cytidine and thymidine (T342C(C−)) in Na+-free, acidified medium (D and E).Immediately adjacent to residue Glyd medium , D and E375 in TM7, mutant S374C(C−) showed a normal uptake profile in Na+-containing, H+-reduced medium but reduced uptake of uridine, cytidine, thymidine, and adenosine in Na+-free, acidified medium (F). Gly340, Gln341, Ser374, and Val375 in hCNT3 correspond to the two adjacent pairs of HP1 and TM7 residues in hCNT1 that have previously been shown to determine the differences in permeant selectivity between that transporter and hCNT2 . Together with previous studies of IH3-TM11 of hCNT3(C−)  and, for comparison, HP1 and TM7 of hCNT1 and HP1 and TM4 of CNT3(C−) , 29, thetopology , all of topology .+-coupled bacterial transporters, including Aquifex aeolicus LeuTAa  revealed multiple examples of PCMBS-sensitive, uridine-protected residue positions with altered Na+-specific hCNT1 and hCNT2, hCNT3 mediates both Na+- and H+-coupled nucleoside cotransport  are clustered together and may involve subdomains within TMs. Most notable in this regard are three residues in TM8 . Other potential conformational differences are even more subtle. The arrows in +-containing, H+-reduced and Na+-free, acidified media but were uridine-protected only in Na+-containing, H+-reduced medium.The present results also contribute insight into cation-dependent conformations adopted by the exofacially facing form of the protein. In contrast to Naransport , 17–20. d one H+ , 20. Thee of Na+ , 20. Pre protein . Buildin protein  and some+-specific E. coli NupC(C−) revealed both similarities and differences between the bacterial transporter and Na+-specific hCNT1 and Na+- and H+-dependent hCNT3.Investigation of HP1 and TM7 of wild-type hCNT1 confirmed the SCAM analysis of the corresponding region of hCNT3(C−) and, in so doing, eliminated any possibility that results for hCNT3(C−) were influenced by removal of endogenous cysteine residues from the transporter. Corresponding SCAM analysis of HP1 and TM4 of H185) reported a H+-dependent conformation of the protein.Although the PCMBS-sensitive residues in NupC(C−) fell within the regions of proposed helical discontinuity in hCNT3(C−) and hCNT1, there were not as many, possibly because of the larger number of functionally inactive mutants that may have an origin in the potential hydrophobic mismatch resulting from insertion of a bacterial protein into a eukaryote membrane environment. Nevertheless, the results provide reinforcing evidence that NupC(C−) HP1 and TM4 contribute to the CNT translocation pore. Within TM4, PCMBS sensitivity of one of the residues . The residues mutated in the present study, or previously, are shown in bold type. Cysteine mutants of each of the four hCNT3 residues potentially involved in Na+ binding were functional and, in three of the four cases, exhibited altered Na+:H+ uridine uptake ratios, being either Na+-preferring  or H+-preferring (Thr370). Two were PCMBS-sensitive (Thr370 and Ile371), one in the presence of H+ only (Thr370). Both of these were uridine-protected.349), and one (Ile350) was inhibited by PCMBS and uridine-protected. In NupC, three of the four corresponding residue mutations were also functional , with one having low but measurable activity . The non-functional residue position was Thr176. Only the Ser142 residue position was inhibited by PCMBS, and this inhibition was not protected by uridine.In hCNT1, all four of the corresponding residue mutations were functional. One had low activity , and three were investigated previously  , the two glutamate substitutions being present at cell surfaces in reduced amounts. Of the three residue positions that retained function following conversion to cysteine , two exhibited altered (Na+-preferring) Na+:H+ uridine uptake ratios (Gln341 and Asn565). All were PCMBS-sensitive under both cation conditions and uridine-protected. Mutation of Gln341 and Thr342 both resulted in changes in permeant selectivity, Gln341 corresponding to hCNT1 Gln320, a residue previously implicated in hCNT1/2 permeant selectivity  . As a grectivity .341, Thr342, and Glu343 yielded proteins with either normal (Gln320) or low but measurable transport activity (Thr321 and Glu322). Similar to hCNT3, all were PCMBS-sensitive and uridine-protected. Likewise, for Na+ binding, all four of the corresponding hCNT1 residue mutations were again functional. One had low activity (Thr349), and one (Ile350) was inhibited by PCMBS and uridine-protected.In hCNT1, mutation of the three residues corresponding to hCNT3 Gln341, Thr342, and Glu343 similarly yielded proteins with either normal (Ser148) or low but measurable transport activity (Gln147 and Glu149). Different from hCNT1/3, however, none were inhibited by PCMBS. Generally, therefore, the present and previous SCAM and other mutagenesis results are consistent with the + and uridine binding.In NupC, mutation of the three residues corresponding to hCNT3 GlnA and C show hCNT3 viewed parallel to the membrane, whereas B and D view hCNT3 from the extracellular surface. Highlighted in yellow in B and D) is the scaffold domain of hCNT3 . Highlighted in blue in B and D) is the corresponding transport domain of hCNT3 comprising two subdomains  and . PCMBS is membrane-impermeable and targets extracellularly accessible residues. As such, side chains of amino acids in red reflect PCMBS-sensitive residues accessible to inhibition in the outward-facing conformation of the transporter (A and B), whereas those in purple show the subset of these residues likely to be within or closely adjacent to the outward-facing permeant binding pocket (C and D). Fig. 10C shows four PCMBS-sensitive and uridine-protected amino acid residues  located deeper and closer to the cytoplasmic side of the membrane but still exposed to the extracellular medium during the transport cycle. Gln341 and Thr342 are located in the flexible, unwound region of the HP1, whereas Asn565 and Ile571 are in the hinge region of TM10, both regions being part of the transport domain for permeant binding.Extending the linear topology model of hCNT3 shown in nsporter , A and Bg pocket , C and D+-coupled transporters are now thought to function in a similar manner (–450 and Leu461), neither of which are uridine-protected, but both of which have locations potentially able to interfere with elevator movement of the transport domain. Similarly, three PCMBS residues  were identified in the hinge region linking TM9 of the scaffold domain to IH3 of the transport domain.Of the 305 residues studied, a total of 53 were PCMBS-sensitive. Of these, 25 were uridine-protected. None of the PCMBS-sensitive residues were located in loops (except that linking TM9 and IH3), and the majority (87%) were located in the transport domain. Overall, the clustering of PCMBS-sensitive residues around the bound uridine molecule suggests that the permeant-binding pockets in the outward- and inward-facing conformations of the transporter are more similar than different. This and the interrelationship between the surrounding outer scaffold and inner transport domains as viewed from the extracellular membrane surface are reminiscent of an emerging novel elevator-type mechanism of transporter function very different from conventional alternating access rocking models of membrane transport. Whereas the latter feature rocker bundle or rocker switch mechanisms to alternately expose bound permeant to extracellular and intracellular membrane surfaces, the elevator mechanism involves a static scaffold domain and a mobile transport domain . To provr manner –49. From manner –. Consistsupplemental Fig. S4). Relative to its inward-facing conformation, the repeat-swap outward-facing model of hCNT3 shows the bound permeant uridine to be elevated and more accessible to the extracellular milieu.A recently developed repeat-swap outward-facing homology model of vcCNT (protein model database (PMDB) identifier PM0080188) is also consistent with an elevator-type transport mechanism . DerivedXenopus oocyte heterologous expression system to undertake PCMBS SCAM analysis of wild-type hCNT1 and E. coli NupC(C−). The results highlight functionally important residues and support the structural topology model for hCNTs predicted from the crystal structure of vcCNT.The present analysis of the IH2 to TM9 region of hCNT3(C−) completes a systematic SCAM analysis of the entire transport domain of the protein. The present study also describes the successful use of the The vcCNT structure is in an inward-facing occluded form, with access to the uridine-binding site completely blocked from the periplasmic surface of the membrane and hindered from the cytoplasmic surface. Transport must therefore involve transitions between this state and conformations in which the binding site is fully exposed either to the periplasmic space or cytoplasm. The specific nature of such transitions remains unclear, although as shown here and previously , 29, a n+-specific and one of which also accepts H+. Given that the vcCNT crystal structure has a single bound Na+, it remains to be determined with which of the two hCNT3 cation-binding sites it is equivalent. Because mutation of hCNT3 residues predicted to be part of that Na+-binding site also showed changes in H+-dependent uridine transport, these residues possibly contribute to the Na+/H+-binding site of hCNT3. On the other hand, other regions of hCNT3 also exhibit residues whose mutation influence both Na+ and H+ coupling  has been previously described. The hCNT3 cDNA provided the template for the construction of a cysteine-less version of hCNT3 (hCNT3(C−)) in which all 14 endogenous cysteine residues were converted to serine (96) was converted to alanine. hCNT3(C−), NupC(C−), and wild-type hCNT1 were transferred into the Xenopus oocyte expression vector pGEM-HE. By providing additional 5′- and 3′-untranslated regions from the Xenopus β-globin gene flanking the multiple cloning site, pGEM-HE gives enhanced production and functional activity of heterologous proteins expressed in Xenopus oocytes  transcription system. Defolliculated stage VI Xenopus oocytes were microinjected with 20 nl of water or 20 nl of water containing capped RNA transcripts (20 ng) and incubated in modified Barth's medium (changed daily) at 18 °C for 72 h prior to assay of transport activity.In hCNT3(C−), NupC(C−), or wild-type hCNT1, residues were individually converted into cysteine using the QuikChange® site-directed mutagenesis kit (Stratagene). Constructs were analyzed in both directions by m NaCl or ChCl, and 2 mm KCl, 1 mm CaCl2, 1 mm MgCl2, and 10 mm HEPES (pH 8.5) or MES (pH 5.5). Uptake was traced with 14C- or 3H-labeled nucleosides   at a concentration of 10 μm for hCNTs or 1 μm for NupC. Transport medium for adenosine uptake experiments also contained 1 μm deoxycoformycin to inhibit adenosine deaminase activity. All uptake values represent initial rates of transport (influx) determined using an incubation period of 1 min for hCNTs and 10 min for NupC (+-free (choline chloride) transport medium , and individual oocytes were dissolved in 1% (w/v) SDS for quantitation of oocyte-associated radioactivity by liquid scintillation counting . Also in a volume of 200 μl, oocytes were treated on ice for 10 min with 200 μm PCMBS. Excess PCMBS was removed by three washes with ice-cold transport medium before the assay of transport activity. As shown in supplemental Fig. S3, a 10-min exposure of wild-type hCNT3 was sufficient to cause maximum inhibition of transport and, as a control, mutant hCNT3(C−) T370C exhibited a similar time course of inhibition (data not shown). In protection experiments, unlabeled uridine (20 mm) was included along with PCMBS  and hCNT3(C−) mutant proteins at oocyte cell surfaces was determined by labeling intact oocytes with EZ-Link sulfo-NHS-LC-biotin (Pierce) followed by isolation of the resulting biotinylated plasma membrane proteins using immobilized streptavidin resin (Pierce) according to the manufacturer's instructions. For immunoblotting, solubilized proteins (one oocyte/lane) were resolved on NuPAGE® Novex® Bis-Tris mini gels (Invitrogen). The electrophoresed proteins were transferred to polyvinylidene difluoride membranes  and probed with affinity-purified anti-hCNT3tibodies . Blots wR. M. conducted the flux and transport inhibition assays and analyzed the results. R. M. and S. Y. M. Y. undertook the surface expression assays, and A. M. L. N. prepared the mutants. C. E. C. and J. D. Y. conceived the project, interpreted the data, and, with R. M. and S. Y. M. Y., wrote the manuscript.