In this study, Th1/Th2 cytokines  profile induced by purified CD4+/CD8+/CD14+ cells were purified from peripheral blood using magnetic beads; CD4+/CD8+ T cells were co-cultured with autologous CD14+ monocytes in the presence of soluble Leishmania antigens (SLA). Stimulation of either CD4+ T cells or CD8+ T cells of HCL volunteers with SLA induced a significantly (P<0.05) higher IFN-γ production compared with the cells of controls. Upregulation of IFN-γ gene expression in CD4+ cells (P<0.001) and CD8+ cells (P = 0.006) of HCL volunteers was significantly more than that of controls. Significantly (P<0.05) higher fold-expression of IFN-γ gene was seen in CD4+ cells than in CD8+ cells. In HCL volunteers a significantly (P = 0.014) higher number of CD4+ cells were positive for intracellular IFN-γ production than CD8+ cells.CD4+ and CD8+ T cells. The results suggested that immune response in protected individuals with a history of zoonotic cutaneous leishmaniasis (ZCL) due to L. major is mediated not only through the expansion of antigen-specific IFN-γ producing CD4+ Th1 cells, but also through IFN-γ producing CD8+ T cells.Collectively, the volunteers have shown maintenance of specific long-term immune responses characterized by a strong reaction to leishmanin skin test and IFN-γ production. The dominant IFN-γ response was the result of expansion of both CD4 Leishmania parasite. Resistance and susceptibility of mice to Leishmania major infection is associated with two types of CD4+ T lymphocytes development: Th1 type response with production of cytokine IFN-γ is associated with resistance, whereas Th2 type response with production of cytokines IL-4 and IL-5 is associated with susceptibility. A clear Th1/Th2 dichotomy similar to murine model is not defined in human leishmaniasis and we need as much information as possible to define marker(s) of protection. We purified CD4+/CD8+ T cells, stimulated them with Leishmania antigens and analysed gene and protein expression of Th1/Th2 cytokines in volunteers with a history of self-healing CL who are presumed to be protected against further Leishmania infection. We have seen significant upregulation of IFN-γ gene expression and high IFN-γ production in the Leishmania stimulated CD4+ T cells and CD8+ T cells. We concluded that both antigen-specific IFN-γ producing CD4+ Th1 cells and IFN-γ producing CD8+ T cells contribute to the long term protection in individuals with a history of CL. This proves the importance of CD8+ T cells as a source of IFN-γ in Th1-like immune responses.Cutaneous leishmaniasis (CL) is usually a self-healing skin lesion caused by different species of  CD8+ T cells are shown to confer a significant role in protection against acute and chronic form of Toxoplasma gondii infection + T cells hamper parasite dissemination by either direct lysis of infected cells or through release of cytokines. During chronic infection CD8+ T cells limit Toxoplasma cyst formation in tissues + T cells, thus vaccines are designed based on induction of infection-blocking CD8+ T cells + T cells are also important in the control of HIV infection + T cells recognize infected cells through an MHC-I dependent process and viral infected cells are lysed by secretion of perforin and granzymes + T cell response against HIV virus, but the response is not enough to successfully control viral replication Listeria monocytogenes infection, both CD4+ and CD8+ T cells contribute in induction of protection, but the major bactericidal role is attributed to CD8+ T cells + T cells, in cooperation with CD4+ T cells, appear to be involved in the induction of host immunity against both primary infection and reinfection of Leishmania parasite L. major infected CD8+ depleted BALB/c mice, during lesion healing the frequency of IFN-γ producing CD4+ T cells and the amount of IFN-γ are diminished resulted in a higher parasite burden L. major induces a transient Th2 type response and then shifts to a Th1 response associated with healing. Induction of this Th1 type of response partly depends on the activation of IFN-γ producing CD8+ T cells and in the absence of CD8+ T cells, the Th2 response is sustained + T cells have been demonstrated to produce IFN-γ and contribute to the rapid healing of secondary lesions which develop after primary challenge with L. majorThere are reports which showed that CD8+ T cells are involved in healing process of CL due to L. braziliensis+ T cells and their cytokines in CL due to L. major.There are reports from New World leishmaniasis which showed that CD8+ cells and CD8+ T cells. In the current study two major lymphocyte subtypes, CD4+ and CD8+ T cells, were purified from individuals with history of self-healing CL and cytokine pattern were analyzed at transcript and protein levels in response to Leishmania antigens.In leishmaniasis, most of the data generated so far is drawn from PBMCs culture without separation of T cell subtypes The study was approved by Ethical Committee of Tehran University of Medical Sciences. Potential candidates were invited and those who were willing to participate and sign a written informed consent were recruited.L. major and with leishmanin skin test (LST) more than zero and as control 18 healthy volunteers from non-endemic area with no response to LST were included. HCL volunteers were selected among the previous Center's patient who received no treatment for the CL lesion(s) and the lesion(s) cured spontaneously within one year of onset. The causative agent of every CL patient was previously identified as L. major using PCR method.Fourteen volunteers with history of self-healing CL (HCL) caused by Leishmania major (MRHO/IR/75/ER) was cultured on NNN medium and passaged on RPMI 1640  supplemented with 10% fetal calf serum (FCS). Promastigotes were harvested at day 5, washed 3 times with PBS (pH 7.2) and used for preparation of soluble Leishmania antigen (SLA) as previously described 9 promastigotes, and then the parasites were freeze-thawed 10 times followed by sonication at 4°C with two 20-sec blasts. Parasite suspension was centrifuged at 30,000×g for 20 min, the supernatant was collected and re-centrifuged at 100,000×g for 4 hours. SLA protein concentration was measured using Bradford method + and CD8+ lymphocytes isolation was performed using magnetic beads system  by positive selection using anti-CD4 or anti-CD8 coated nanoparticles. Briefly, cell suspension was prepared at a concentration of 1×107 cells/ml in a 5 ml tube in isolation buffer containing PBS plus 2% (v/v) FBS and 1 mM EDTA. EasySep CD4/CD8 cocktail Abs was added at 10 µl/ml cells, mixed well and incubated at room temperature (RT) for 15 min. Magnetic nanoparticles were added at 5 µl/ml cells and incubated for 10 min at RT. The cell suspension was brought to 2.5 ml by adding buffer and the tube was placed into the magnet for 5 min, then the supernatant was discarded. The desired cells were remained bound inside the tube. The steps of placing tube into the magnet were repeated three times.Twenty mL of blood sample was collected from each volunteer and Peripheral Blood Mononuclear Cells (PBMCs) were isolated using Ficoll–Hypaque  density gradient centrifugation. CD4+) were isolated from autologous PBMC by negative selection according to the manufacturer's instruction . Briefly, cell suspension was prepared at a concentration of 5×106 cells/ml in isolation buffer. EasySep monocyte enrichment cocktail Abs was added at 5 µl/ml cells, mixed well and incubated at 4°C for 10 min. Magnetic microparticles were added at 5 µl/ml cells for 5 min at 4°C. The cell suspension was brought to 2.5 ml by adding buffer and the tube was placed into the magnet, for 2.5 min at RT. The desired unbound fraction was transferred into a new tube.Monocytes  following mitomycin C  treatment at a final concentration of 10 µg/ml for 30 min at 37°C with 5% CO2.+ or CD8+ lymphocytes were adjusted to 0.5–1×106 cells/ml mixed with 1∶10 of autologous monocytes and were cultured in U-bottomed 96-well plates  in the presence of either 10 µg/ml PHA or 50 µg/ml of SLA in a final volume of 200 µl. Plates were incubated at 37°C with 5% CO2 in humidified atmosphere for 72 hrs.The cells were cultured in RPMI 1640 media supplemented with 10% heat-inactivated human AB Rh+ serum, 10 mM/L Hepes, 2 mM L-glutamine, 100 U/ml penicillin G and 100 µg/ml streptomycin . CD4Culture supernatants were collected at 72 hours, the level of IL-5, IL-10, IL-13 and IFN-γ were titrated in culture supernatants using ELISA method . Briefly, the plates were coated with anti-IFN-γ/IL-5/IL-10/IL-13 mAb in PBS, pH 7.4, and incubated at 4°C over night. After blocking the wells using buffer containing PBS plus 0.05% (v/v) Tween 20 and 0.1% (w/v) BSA, supernatants were added to each well. Biotin-labeled mAb in incubation buffer was added to each well and as enzyme streptavidin-HRP was used. The reaction was developed using 3,3′,5,5′-tetramethylbenzidine (TMB) substrate and stopped with 0.5M H2SO4 solution. The plates were washed after each step of incubation using PBS+0.05% (v/v) Tween20. The plates were read at 450 nm using a reader . The mean optical densities (ODs) of triplicate cultures were compared with the standard curves prepared using recombinant IL-5, IL-10, IL-13 and IFN-γ. The cytokine levels represent the differences between the ODs of test and background wells. The detection limit of the assays was 4 pg/ml for IL-5 and 0.5 pg/ml for IL-10, 5 pg/ml for IL-13 and 2 pg/ml for IFN-γ.5 per ml and stimulated with PMA  50 ng/ml plus Ionomycin calcium (Sigma) 500 ng/ml and incubated at 37°C, 5% CO2 for 5–6 hrs. Monensin (Sigma) was added at 25 µM/ml during the last 4–5 hrs of culture for blocking. Cells were harvested and washed 2 times with PBS (pH 7.2) plus 0.1% bovine serum albumin (BSA). The cells were permeabilized using BD Cytofix/Cytoperm kit according to the manufacturer's instruction . In the final step, cells were stained with FITC-conjugated mouse anti-human IFN-γ and PE-conjugated rat anti-human IL-2 . Cells were washed ×2 with perm/wash buffer and resuspended in PBS (pH 7.2) plus 1% BSA. Cells were analyzed using Partec flow cytometer  while isotype matched negative controls were used to set the threshold of autofluorescence. A minimum of 50,000 events were acquired for each sample. FACS data analysis was performed using FloMax (DAKO cytomation) software.After SLA stimulation, part of the cells was used for ICS assay. Cells were adjusted at 5×10At the time of supernatants collection (at day 3), the SLA stimulated cells were harvested and used for RNA extraction. The procedure began with reverse transcription of mRNA to cDNA. The cDNA was then used as template for Real-time PCR using specific primer of each cytokine. Solutions were treated and glassware was filled with 0.1% (v/v) diethylpyrocarbonate (DEPC)  in H2O.6 cells. RNA was solublized through pipetting and incubated at RT for 5 min. Then 0.2 ml chloroform was added per 1 ml of homogenate, the suspension was shook vigorously and kept on ice for 5 min followed by centrifugation for 15 min, 12,000×g at 4°C. The upper phase was collected and added to an equal volume of isopropanol and incubated at 4°C over night. Then the cell suspension was centrifuged at 12,000×g, 4°C for 15 min, the supernatant was discarded and the RNA pellet was washed with 1 ml 75% ethanol at 7,500×g for 8 min. At the end, the pellet was drayed briefly and dissolved in DEPC treated water. The purity of RNA samples was assessed by the ratio of ODs at 260/280 nm using UV spectrophotometry.The cell pellet was resuspended in cold PBS (pH 7.2) and lysed by addition of 0.2 mL of Trizol  per 1×1018 primer was added to about 3 µg of total RNA, mixed and incubated at 70°C for 5–10 min. The tube was placed on ice for a few minutes, centrifuged briefly and added with: 4 µL of 5× reaction buffer, 1 µL 10 mM dNTPs, 20 u RNase inhibitor  and DEPC treated water up to 30 µL. Tube was incubated at 37°C for 5 min, the contents were mixed gently and then added with 100 U of enzyme. The reaction mixture was incubated at 42°C for 60 min. The enzyme was inactivated by heating at 70°C for 10 min. and chilled on ice. In a Real-time PCR MicroAmp optical 96-well reaction plate  for 25 µl reaction mixture the followings in each optical well were prepared: 12.5 µl QuantiTect SYBR Green I , 3 µl cDNA, 2 µl primer pair mix (0.5 µM each), 7.5 µl dH2O  in a 30 µL reaction mixture. Briefly, 1 µL (0.5 µg) of oligo dTFor normalizing the difference in the amount of inputting cDNAs, the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal standard. Samples and internal standard were amplified in separate wells of the plate. Two-step thermal profile as a PCR program was set up on the SDS software (version 1.3.1) of Applied Biosystems 7,500 machine .−ΔΔCt method was used to calculate relative changes in the gene expressions:The Ct (threshold cycle) of each sample was used in gene relative expression calculation. The 2−ΔΔCt, the amplification efficiencies of the target and reference (GAPDH) genes must be approximately equal. For this purpose, the Ct values variation with cDNA template dilutions was checked. A pooled cDNA preparation was diluted over a 10-fold range and PCR was performed for each dilution using specific primers. A plot of the log cDNA dilution versus CT was prepared. The slope of each line was obtained from regression equation and the efficiencies of the target and reference genes were calculated using the equation: Efficiency (E) = [10 (1/slop)]−1To have a valid calculation of 2Non-parametric tests of Mann-Whitney, Kruskal-Wallis and Dunn's post-test for paired comparisons were used for statistical analysis of the data using SPSS version 11.5  and GraphPad Prism version 5.01  softwares. Nonparametric tests were chosen because the samples did not follow a Gaussian distribution. P value of <0.05 considered to be significant.Basic information of the studied population is outlined in +, CD8+ T cells culture. The amount of IFN-γ level was significantly higher in PBMC culture of HCL volunteers compared with that of healthy controls (P<0.05). Results of purified T cell culture showed that stimulated CD4+ T cells from HCL volunteers induced a significantly higher IFN-γ production compared with cells from healthy controls (P<0.05)   were measured on supernatants collected at 72 hrs of SLA stimulated PBMC or CD4(P<0.05) . Similar(P<0.05) . The levcontrols . IL-5 le−ΔΔCT method. The expression amount was compared with unstimulated cells of culture and relative fold-expression was reported. Result for each donor was calculated and then data was pooled and presented as a mean of HCL volunteers against healthy controls. Results showed that the upregulation of IFN-γ gene expression in CD4+ cells from HCL volunteers was significantly higher than that of healthy controls (P<0.001)  . Ct values of amplified templates of antigenic stimulated T cells were used for calculation of different cytokine gene expressions using 2P<0.001) . Similar= 0.006) . Compari+ and CD8+ T cell cultures, the changes in the gene expression of IL-5, IL-10 and IL-13 were not significantly different between HCL volunteers and healthy controls.In both CD4By amplification of serially diluted pooled cDNA, the amplification efficiency of the target (cytokines) compared to reference (GAPDH) genes was examined using SYBR Green detection. Using the equation pointed out in methods, efficiency of GAPDH was 96% while that of targets were between 91% and 95%.+/CD8+ T cells, part of the cells were harvested and stimulated with PMA plus Ionomycin for 5–6 hrs, stained for intracellular IFN-γ with conjugated mAbs and the frequency of positive cells was analyzed using flow cytometry. In CD8+ cells compartment, antibody to CD56 marker allowed to exclude IFN-γ positive populations of natural killer cell sources (+ and CD8+ T cells (+ T cells were positive for intracellular IFN-γ production than CD8+ cells (P = 0.014).Seventy two hrs after SLA stimulation of CD4 sources . Results T cells . Based oL. major infection in murine model depend upon induction of Th1 or Th2 response, respectively in vivo LST and in vitro lymphocyte response to Leishmania antigens + T cell and CD8+ population and there is no report to show a clear-cut CD4+ Th1/Th2 response + Th1/Th2 and CD8+ T cells in volunteers recovered from CL is evaluated at the transcript and protein levels.Resistance and susceptibility to Leishmania antigens (SLA) stimulated cells showed that pure CD4+ T cells induced a significantly higher IFN-γ production in HCL volunteers compared to that of healthy controls, IL-5 as a Th2 type cytokine was not detectable and IL-10 and IL-13 levels were not significantly different in culture of T cells from HCL volunteers compared with that of healthy controls. At the same time, the level of cytokines' mRNA expression was evaluated to detect T cell cytokine responses to Leishmania stimulation at transcript level, after several experiments to explore the optimum stimulation time points for mRNA analysis. Simultaneous analysis of cytokines gene expression showed a strong up-regulation of Th1 cytokine IFN-γ mRNA in CD4+ T cells. In line with the results of secreted proteins, level of Th2 cytokines transcripts including IL-5, IL-10 and IL-13 showed no significant increase in HCL volunteers compared with healthy control. The up-regulation of IFN-γ transcripts expression in Leishmania stimulated CD4+ T cells consistent with IFN-γ secretion is an indication of Th1 type of response in HCL volunteers parallel with no Th2 response indicating by low level of IL-5, IL-10 and IL-13 cytokines.The results of soluble + T cells showed also a significantly higher IFN-γ production in HCL volunteers compared to the healthy control volunteers. Similar to CD4+ T cells, the levels of IL-10 and IL-13 were not significantly different in CD8+ T cell culture between HCL volunteers and healthy controls. When CD4+ and CD8+ T cell response was compared, the level of IFN-γ secretion in SLA stimulated cells was not significantly different between CD4+ and CD8+ cells, but real-time PCR analysis revealed that expression level of IFN-γ mRNA was higher in CD4+ T cells than CD8+ T cells. To confirm the real-time PCR results, part of CD4+/CD8+ sorted T cells were harvested following SLA stimulation and intracellular production of IFN-γ was assessed using flow cytometry. Results of intracellular cytokine staining (ICS) in HCL volunteers confirmed that a significantly higher number of CD4+ T cells produced intracellular IFN-γ compared with CD8+ T cells (median = 15% vs. 8%). Based on the results it seems that the source of IFN-γ production is both CD4+ Th1 cells and CD8+ cells in individuals with history of CL.Analysis of cytokine secretion and transcript expression of SLA stimulated CD8+ T cells in human Leishmania infection is not well known and existed reports are controversial. In a study performed on Sudanese individuals it was suggested that IFN-γ production is associated with CD4+ T cells rather than CD8+ T cells in individuals with history of CL due to L. majorL. braziliensis, a higher proportions of CD4+ than CD8+ T cells was present L. braziliensis, the frequency of CD4+ and CD8+ T cells was the same with approximately constant production of IFN-γ Leishmania specific CD8+ T cells in the lesions and peripheral blood during acute phase and healing process in L. major or L. braziliensis CL patients + T cells for the control of L. major infection is shown to be partly dependent on the procedure of challenge: β2-microglobuine or CD8+ deficient C57BL/6 mice when challenged with high dose of L. major have the ability to cure the lesion, which indicates that CD8+ T cells are not necessary for the control of primary L. major infection in anti-CD8 Ab treated or CD8 deficient mice was dependent on the CD8+ T cells + T cells was studied in CBA and anti-CD4 mAb treated BALB/c mice healed from L. major infection. The cured mice were re-challenged with L. major in the contralateral footpad and lymph nodes cells were depleted of CD4+ T cells and stimulated in vitro. The remaining CD8+ T cells produced a significant amount of IFN-γ Leishmania infection and induction of protection in murine model CD8+ T cells play an important role The role of CD8in vitro restimulated T cells to check the type of immune response elicited against Leishmania antigen. The main cytokine produced was found to be IFN-γ in the volunteers' T cells. IFN-γ eliminates intracellular pathogens primarily through macrophage activation. Macrophages upon activation produce nitric oxide (NO) which inhibits growth of intracellular pathogens. It is shown that during active lesion of CL due to L. major the proliferative response and IFN-γ production of PBMC was increased L. braziliensis infection demonstrated a Th1/Th2 mixed response in early stage of active CL L. major reinfection was assessed in mice. The neutralization of IFN-γ at the time of reinfection reduced the specific DTH response, showing the involvement of IFN-γ in the recall of memory response to L. majorT. gondii, it is shown that CD8+ T cells confers resistance against acute infection + T cells play a significant role in controlling chronic T. gondii infection and inhibits encephalitis in mouse model In the present study, following the isolation of CD4+/CD8+ T cells, Th1/Th2 cytokines were titrated on culture supernatant of + cells was less than CD4+ cells, but interestingly a significant amount of IFN-γ was produced by CD8+ T cells in cell culture and around 5–12% of CD8+ cells was positive for IFN-γ secretion by ICS assay. It is concluded that CD8+ T cells contribute along with CD4+ Th1 cells in IFN-γ production in individuals with history of CL. Despite the limited reports of CD4+ Th1 cells as the main source of IFN-γ production in CL patients + Th1/Th2 paradigm in human CL, PBMCs rather than purified T cells were used, hence the role of IFN-γ producing CD8+ T cells should not be ruled out when reporting a “Th1” type response in PBMC culture.In the current study, even though using real-time PCR the expression level of IFN-γ transcripts in CD8L. braziliensis is previously shown Leishmania infection. The blood samples were collected a few months to years after cure of CL lesions. The strong LST response (mean LST = 10.7±7.5 mm) and IFN-γ production is an indication of sustaining cell mediated immune response. This sustaining protective immunity is mediated not only through the expansion of antigen-specific IFN-γ producing CD4+ Th1 cells, but also through IFN-γ producing CD8+ T cells. The question that which one of these T cell subsets plays a more important role in IFN-γ production at the initiation of exposure to sand fly bite needs to be explored.The strong lymphoproliferative and IFN-γ response in self healing CL caused by


The small overlapping intervals facilitated the delineation of two contiguous minimally deleted regions of 0.76 Mb, defined by D1S468 and D1S2845, and of 0.41 Mb, bound by D1S2893 and D1S1608, on 1p36.31–36.32. Based on current reference human genome sequence these deletion regions have been sequenced almost to entirety and contain eight annotated genes. TP73, DFFB and SHREW1 are the only known genes located in these deletion regions, while the others are uncharacterised novel genes. In conclusion, our study has narrowed down the critical tumour suppressor loci on 1p36.3, in which two minimally deleted regions are mapped, and markedly reduced the number of candidate genes to be screened for their involvement in OT development.Loss of the short arm of chromosome 1 is a hallmark of oligodendroglial tumours (OTs). Deletion mapping studies in OTs have revealed multiple commonly deleted regions on chromosome 1p, suggesting that there are more than one tumour suppressor gene. To map critical deletion regions on 1p, a series of 25 OTs were examined for loss of heterozygosity (LOH) on 19 polymorphic markers across the 1p arm using microsatellite analysis. Our study revealed that 60% of tumours had LOH of all informative markers on 1p and identified one tumour showing LOH at telomeric markers only. Since this deletion region lies in one of the critical deletion intervals defined previously, we then screened another series of 27 OTs specifically at 1p36.3 for LOH using nine polymorphic markers. A total of 12% (six out of 52) of tumours were found to carry interstitial deletions. The allelic status and the deletion breakpoints in these tumours with interstitial deletion were further verified by fluorescent  Oligodendroglial tumours (OTs) are primary brain neoplasms that constitute about 5–18% of all gliomas. These tumours comprise the classic oligodendrogliomas and mixed oligoastrocytomas. Two malignancy grades are recognised by the World Health Organization (WHO): grade II for the well-differentiated tumours and grade III for the anaplastic variants , 14 anaplastic oligodendrogliomas (WHO grade III), 13 oligoastrocytomas (WHO grade II) and three anaplastic oligoastrocytomas (WHO grade III), and their corresponding blood samples were obtained from Prince of Wales Hospital, Hong Kong and Hua Shan Hospital, Shanghai, China. The OTs were classified according to the current WHO Classification of Tumours of Nervous System , 50 mM KCl, 2.5 mM MgCl2, 0.2 mM deoxyribonucleoside triphosphates, 0.5 U of AmpliTaq Gold DNA polymerase , and 0.4 μM of each primer. The PCR commenced with an enzyme activation step at 95°C for 10 min, 40 cycles of 94°C for 1 min, 55–60°C for 1 min and 72°C for 1 min, and finalised with an elongation step at 72°C for 10 min. PCR products of multiple markers were pooled and electrophoresed in denaturing 4% polyacrylamide gels using an ABI Prism 377 automated DNA sequencer (Applied Biosystems). The data collected were analysed using GeneScan Analysis software version 3.1 (Applied Biosystems). Allelic imbalance was defined by calculating the allelic ratio of both normal (N) and tumour (T) DNA, based on the formula of (N2/N1)/(T2/T1), where N2 or T2 represents the peak height of the longer allele and N1 or T1 represents the peak height of the shorter allele. LOH was inferred when the allelic ratio was greater than 1.5 or smaller than 0.5, representing loss of the shorter or longer allele, respectively.A total of 30 microsatellite loci, located on chromosome arms 1p, 1q and 19q, were investigated for loss of heterozygosity (LOH) according to reported protocol  for 20–30 min, rinsed in phosphate-buffered saline and dehydrated. The sections were then treated with microwave (600 W) in citrate buffer (pH 6.0) for 10 min, baked at 80°C for 30 min and subjected to hybridisation.Dual-colour FISH analysis was performed on formalin-fixed paraffin-embedded tissue sections as reported with modification (M Tris-HCl (pH 7.5), 10 mM MgCl2, 0.01% bovine serum albumin, 0.1 mM dATP, 0.1 mM dCTP, 0.1 mM dGTP, 0.1 nM dTTP, 0.1 nMβ-mercaptoethanol, 0.03 U DNase I, 20 U of DNA polymerase I, and 0.85 nmol of digoxigenin-1-dUTP (for target probe) or 1.9 nmol of biotin-16-dUTP (for reference probe)  at 15°C for 25–45 min. The labelled probes were mixed with fish sperm and Cot-1 DNA in Hybrisol VI solution , denatured and applied onto sections. Hybridisation was carried out at 37°C overnight. Sections were washed with 1.5 M urea/1 × saline sodium citrate (SSC) at 45°C for 30 min, 2 × SSC at 45°C for 15 min, treated with either antidigoxigenin-rhodamine antibody (Sigma) or avidin conjugated-fluorescein isothiocyanate  at 37°C for 1.5 h, washed in phosphate-buffered saline detergent (Appligene Oncor), and counterstained with Vectashield mounting medium with anti-fade solution of 4,6-diamidino-2phenylindole .Seven bacterial artificial chromosomes (BACs) or bacteriophage P1-derived artificial chromosomes (PACs) were labelled to generate locus-specific FISH probes. These included six target probes for 1p arm and one reference probe for 1q32 (BAC clone RP11-79I9 encompassing the marker STSG28964). The genomic clones were purchased from Invitrogen Corporation . DNA was labelled by nick-translation in a reaction containing 100 mSections were viewed under a Zeiss Axioplan microscope and images of rhodamine, FITC and DAPI were captured through a triple-pass filter by a cooled charge-coupled device. A total of 200 nonoverlapping tumour nuclei were counted on each section. A sample was considered having deletion of 1p locus when more than 70% of counted nuclei exhibited one target signal (red) and two reference signals (green).χ2-test or Fisher's exact test, whichever was appropriate. An obtained P-value less than 0.05 (two-sided) was considered statistical significant.Statistical analysis was performed using the software SPSS 10.0. The correlation between two parameters was evaluated by To localise tumour suppressor loci on the short arm of chromosome 1 in OTs, we carried out a deletion mapping study across the entire 1p arm using microsatellite analysis. A series of 25 OTs were investigated for LOH at 19 microsatellite loci, spacing at an average interval of 7 Mb on the 1p arm. Each tumour showed informativeness in at least seven markers. Overall, 15 (60%) tumours demonstrated LOH in at least one marker, with 14 of them showing LOH at all informative markers tested suggesting that these tumours had lost a copy of the entire or most of 1p arm. One tumour (A18) showed LOH at D1S468 and its juxtaposed marker D1S2795, both of which map to the telomeric end of 1p36.3, but retained heterozygosity at all informative markers centromeric to D1S2795 , for which tissues were available, were subjected to FISH. Representative results of FISH are shown in Figure 3Based on the latest version of reference human genome sequence  the MDRs are covered by two sequence contigs (NT_004321 and NT_004547) with a gap of suggested size of 50 kb between these contigs. MDR1 starts from D1S468, which locates about 230 kb to the proximal end of NT_004321, spans over the sequence gap and ends at D1S2845, which is 480 kb from the distal end of NT_004547, whereas MDR2 lies in NT_004547. There are seven annotated genes clustered at the distal region of MDR1 on NT_004321  and D1S2625 (1q31.2), were investigated for LOH on 1q arm. All tumours except case B24 were informative at either or both markers. Three tumours  had lost heterozygosity at both markers on 1q and also at all informative markers on 1p, suggesting that the entire chromosome was lost. Allelic loss of both 1q markers was also observed in tumour A19, which harboured interstitial deletion of 1p at D1S2845. B4 had lost heterozygosity at distal marker D1S2625 but maintained allelic balance at D1S215, whereas A25 showed LOH at D1S2625 and was noninformative at D1S215. Together, six out of 51 (12%) informative OTs exhibited LOH on 1q.Two markers, D19S219 and D19S412, which are 1.02 Mb apart and locate in the common region of deletion on 19q13.3 were investigated for allelic loss. Out of 51, 33 (65%) informative tumours exhibited LOH in at least one of the 19q markers. In all, 10 tumours  had lost heterozygosity at either marker but maintained allelic balance at the other marker on 19q.Taken together, 29 out of 51 (57%) informative tumours exhibited LOH of at least one marker on both 1p and 19q. Three tumours  had 1p LOH in the absence of detectable 19q LOH, whereas four tumours  exhibited 19q LOH but no 1p LOH.P<0.0001). No statistical significance was observed between 1p LOH, 19q LOH or combined 1p/19q LOH and patient's gender, tumour type or grade. All six OTs with interstitial deletion on 1p36.3 were of WHO grade II, but only a trend was seen between interstitial deletion and tumour grade (P=0.065).The 1p and 19q allelic status was correlated with clinicopathological parameters. 1p LOH was found to be closely associated with 19q LOH in OTs (Recurrent deletion of chromosome 1p in OTs strongly implies the presence of tumour suppressor gene(s) on this chromosome arm. With an aim to isolate the tumour suppressor gene by positional cloning approach, we set out to map the minimally deleted regions on the 1p arm. A series of 52 OTs were examined for allelic deletion by two rounds of microsatellite analysis followed by confirmation with FISH. Six tumours were found to harbour small interstitial deletion specifically on 1p36.3. Previous deletion mapping studies had revealed a few OTs with interstitial deletion on 1p, thus hindering a precise mapping of the critical deletion region . Of thesin vitro (The TP73 gene encodes product that shares significant structural and functional homology with the p53 tumour suppressor, raising the possibility that p73 might be a putative tumour suppressor (reviewed in in vivo. Upon apoptosis induction, caspase cleaves the inhibitor and releases DFFB from the complex to enter into nuclei to degrade chromosomal DNA (DFFB is the other known gene identified in MDR1. It encodes the caspase-activated DNase and is responsible for degradation of chromosomal DNA during apoptosis. Its gene product is complexed with an inhibitor encoded by DFFA β-catenin. However, in nonpolarised invasive epithelial cells, shrew1 bound neither to N-cadherin and β-catenin (β-catenin are detectable at cell–cell junctions in malignant astrocytic tumours (β-catenin status in OT is not known. Further investigation of SHREW1 function may shed light on the role of this gene plays in cancer development and its involvement in invasion.SHREW1 appears to be the single gene located in MDR2. Recently, the cDNA of SHREW1 was isolated from invasive endometriotic cells (In conclusion, our deletion mapping study has refined one of the candidate tumour suppressor loci on chromosome arms 1p in OTs. Two contiguous MDRs are delineated from a small interval on 1p36.3, between D1S468 and D1S1608, which contain only eight known and novel genes. These candidate genes are currently under investigation for their involvement in OTs.


Human RNase P has been initially described as a tRNA processing enzyme, consisting of H1 RNA and at least ten distinct protein subunits. Recent findings, however, indicate that this catalytic ribonucleoprotein is also required for transcription of small noncoding RNA genes by RNA polymerase III (Pol III). Notably, subunits of human RNase P are localized in the nucleolus, thus raising the possibility that this ribonucleoprotein complex is implicated in transcription of rRNA genes by Pol I.By using biochemical and reverse genetic means we show here that human RNase P is required for efficient transcription of rDNA by Pol I. Thus, inactivation of RNase P by targeting its protein subunits for destruction by RNA interference or its H1 RNA moiety for specific cleavage causes marked reduction in transcription of rDNA by Pol I. However, RNase P restores Pol I transcription in a defined reconstitution system. Nuclear run on assays reveal that inactivation of RNase P reduces the level of nascent transcription by Pol I, and more considerably that of Pol III. Moreover, RNase P copurifies and associates with components of Pol I and its transcription factors and binds to chromatin of the promoter and coding region of rDNA. Strikingly, RNase P detaches from transcriptionally inactive rDNA in mitosis and reassociates with it at G1 phase through a dynamic and stepwise assembly process that is correlated with renewal of transcription.Our findings reveal that RNase P activates transcription of rDNA by Pol I through a novel assembly process and that this catalytic ribonucleoprotein determines the transcription output of Pol I and Pol III, two functionally coordinated transcription machineries. Transcription is carried out by functionally distinct nuclear RNA polymerases (pols) associated with general transcription factors, as well as specificity and coregulatory factors that assist in formation and function of preinitiation complexes. Pol I transcribes rRNA genes, Pol II mainly synthesizes protein-coding genes, while Pol III transcribes a large set of small noncoding RNA genes. Recent findings reveal that noncoding RNAs associate with and regulate pols I, II and III Human nuclear RNase P has been initially characterized as a tRNA processing ribonucleoprotein, consisting of H1 RNA and at least ten distinct protein subunits, termed Rpp14, Rpp20, Rpp21, Rpp25, Rpp29, Rpp30, Rpp38, Rpp40, hPop1 and hPop5 We have previously demonstrated that human nuclear RNase P is required for transcription of small noncoding RNA genes transcribed by Pol III H1 RNA is an abundant molecule in the cell. This transcript was detected in the cytoplasm, nucleoplasm and nucleoli. Protein subunits of human RNase P have also been differentially detected in specialized intranuclear compartments associated with active gene transcription, including nucleoli In this study, we show by biochemical and reverse genetic means that H1 RNA and its protein subunits, as part of an RNase P ribonucleoprotein, are required for efficient transcription of rDNA by Pol I. RNase P copurifies and associates with components of Pol I and its transcription initiation factors and exerts its role in transcription through association with the promoter and coding region of rDNA. Furthermore, we demonstrate that RNase P disengages from rDNA in mitosis and reassociates with it at G1 phase through a dynamic and stepwise assembly process. Our data implicate a catalytic ribonucleoprotein in transcription by Pol I and Pol III, whose coordinated function is critical for protein synthesis and cell growth.Rpp29 was targeted for destruction in HeLa cells by the use of RNA interference. Western blot analysis revealed efficient knockdown of Rpp29 in cells transfected with siRNA29 Rpp21, when compared with control cells , and thus exhibited reduced activity of Pol I . This reThe findings described above provide evidence that a protein subunit of human RNase P can substitute for its endogenous counterpart by reconstituting transcription of Pol I and Pol III.Tyr by RNase P  murine Pol I holoenzyme with its transcription factors has been reported The above findings demonstrate that an active RNase P associates with components of Pol I, which seems to exist in a large complex with its transcription initiation factors. The purification of a large  analysis see [10]. Gennd Rpp29  but not nd Rpp29 , consistnd Rpp29 , while tnd Rpp29 . Chromatnd Rpp29 , while tnd Rpp29 . Hence, We next scanned the human rDNA transcription units (each is ∼14 Kbp) for binding of protein subunits of RNase P by ChIP analysis using specific primers that correspond to distant regions within these units see . In thesTranscription of rRNA genes is regulated during the cell cycle, whereby it gradually increases from G1 to S phase, peaks at G2 and ceases at mitosis Leu gene until mid G1 , which coincides with increased transcription by Pol I. Accordingly, Rpp25 seems to contribute to the elevated activity of Pol I at S phase (or late G1), a conclusion that is supported by the finding that recombinant Rpp25 stimulates transcription by Pol I in whole cell extracts lacking endogenous Rpp25. Similarly, recruitment of RNase P to 5S rRNA and tRNA genes after mitosis proceeds via a dynamic and stepwise association process of its subunits.A line of evidence supports the prospect that protein subunits of human RNase P could be recruited as independent entities to chromatin of target genes. Thus, RNA interference shows that knockdown of one protein subunit does not necessarily obstruct the recruitment of other subunits on target genes in cycling cells, even though transcription is inhibited. For example, knockdown of Rpp25 did not lead to disengagement of Rpp20 and Rpp29 from rDNA  and knocFinally, binding of a large ribonucleoprotein complex, such as human RNase P with its two Alba-like proteins, Rpp20 and Rpp25, to chromatin of hundreds of genes transcribed by Pol I and Pol III should have global effects on the structure, spatial organization and function of the genome 2 at 37°C. For transfection, 1–5×105 cells grown in 92×17 mm style petri dishes were transfected with the desired siRNA (15–30 µg/dish) or plasmid DNA (15 µg) in 10 ml medium using the calcium phosphate method. For knockdown of Rpp21 and Rpp25, plasmids (10–15 µg) carrying siRNA-coding genes (kind gifts of Prof. Sidney Altman)Adherent HeLa cells were grown in high glucose DMEM (Invitrogen) supplemented with 5% fetal bovine serum, streptomycin (100 µg/ml), penicillin (100 U/ml) and Nystatin (12.5 U/ml). Cells were incubated in 5% COFor synchronization, HeLa cells were grown in 92×17 mm style petri dishes for 50% confluence and then treated with nocodazole (40 ng/ml) for 16 h. The dishes were shaken to detach mitotic cells from G2-enriched, adhered cells as described by White et al. 1995 Reverse transcription was performed using M-MLV reverse transcriptase (Promega) with equal amounts of total RNA (1–2 µg) extracted from treated HeLa cells and 5–10 pmol of gene-specific reverse primers (see below). Amplification of the 5′ETS sequence of the reverse-transcribed pre-rRNA by PCR was performed as described by others 2 and 1 mM DTT. RNase P was then eluted from the column using a 100–500 mM KCl gradient 32P-labeled E. coli precursor tRNATyr. RNase P activity is eluted at 250–300 mM KCl 2r = 1×105) in the void volume.Rapidly dividing HeLa cells (at ∼50% confluence) in sixty 175-cm square flasks were pelleted, disrupted, and the cell homogenate was centrifuged at 7,000 rpm followed by another centrifugation at 31,000 rpm in a Beckman Ti60 rotor to obtain S100 crude extract 2, 80 mM KCl, 0.5 mM DTT, 10 mM creatine phosphate), NTPs , 5 µCi of [α-32P]-UTP  and 0.25 µg of plasmid carrying the mini-rDNA gene. After 1 h of incubation at 30°C, reaction mixtures were diluted 1∶1 with H2O, passed through a G-50 column, diluted to 250 µl with 1× digestion buffer , and digested with 120 µg/ml Proteinase K for 30 min at 37°C. RNA was recovered following phenol∶chloroform∶isoamylalcohol extraction by ethanol precipitation and labeled RNAs were analyzed in 8% polyacrylamide gels. Bands were visualized by autoradiography and quantitated by Scion Image software.In vitro transcription reactions of the mini-rRNA gene in newly prepared whole HeLa extracts were performed in a final volume of 25 µl that contained 15 µl of extract, 1× transcription buffer .I110, UBF, as described 32P-labeled precursor tRNATyr in 1× PA buffer mixed with 1× TNET buffer containing 2 mM Tris-HCl, pH 7.5, 35 mM NaCl, 0.1 mM Na2EDTA, 1 mM 2-mercaptoethanol, 0.01% Triton X-100, 4 U of rRNasin and 12 µg Poly I∶C. Cleavage products were separated in 8% polyacrylamide/7 M urea gel.RNase P from whole HeLa extracts was immunoprecipitated with polyclonal anti-Rpp antibodies or with monoclonal and polyclonal antibodies directed against Pol I, TAFChIP analysis was done essentially as previously described Sonicated material (0.2 ml per each immunoprecipitation) was precleared for 30 min with 20 µl of Protein A/G Plus agarose beads (Santa Cruz Biotechnology) and then blocked with 2 µg of salmon sperm (Invitrogen). After centrifugation, the sample was subjected to IP overnight at 4°C using a nutating device with the appropriate antibody coupled to beads (coupling was for 6 h at 4°C) and in the presence of 4 µg of salmon sperm. Precipitated complexes were washed three times with 1 ml of RIPA buffer , three times with 1 ml of LiCl buffer , and three times with 1 ml of TE. Immunoprecipitated material was eluted twice with 200 µl of 1% SDS/TE and incubated overnight at 42°C in 0.4 ml volume of elution buffer containing 120 µg/ml Proteinase K. DNA was extracted twice with phenol/chloroform/isoamyl alcohol (25∶24∶1 v/v), ethanol-precipitated, washed with 70% ethanol, dried, and resuspended in 25 µL distilled water for use in PCR analysis.Tyr, U1 snRNA, ARPP P0 were described 5′- CGACTCTTAGCGGTGGATCAC-3′ and 5′-AAGCGACGCTCAGACAGGCGT-3′, for 18S rDNA: 5′-CCTTTAACGAGGATCCATTGGA-3′ and 5′-GACACTCAGCTAAGA GCATCGAG -3′, for 28S rDNA: 5′-CTCTTCCTATCATTGTGAAGCAG-3′ and 5′-CAAATGTCTGAACCTGCGGTTC-3′ and for IGS: 5′-TGTTCTTGGGGGTGGGTTGAC-3′ and 5′-GAAGAGGTTCCGATGGGAAGTTG-3′.Each PCR reaction contained 2.5 µL of each primer , 5 µl of 10× reaction mixture, and 4 µl of DNA for each IP sample. Input DNA was diluted in distilled water before PCR. PCR amplification programs and primer sequences for 5S rRNA, tRNA32P-labeled E. coli precursor tRNATyr and separation of the cleavage products in an 8% sequencing gel. An aliquot of 15 µl from each treated extract was then tested for in vitro transcription of mini-rRNA and small noncoding RNA genes as described above.Targeting the H1 RNA for specific cleavage by RNase H has been previously described


Diacylglycerol (DG) kinase (DGK) phosphorylates DG to produce phosphatidic acid (PA). Of the 10 subtypes of mammalian DGKs, DGKβ is a membrane-localized subtype and abundantly expressed in the cerebral cortex, hippocampus, and caudate-putamen. However, its physiological roles in neurons and higher brain function have not been elucidated.We, therefore, developed DGKβ KO mice using the Sleeping Beauty transposon system, and found that its long-term potentiation in the hippocampal CA1 region was reduced, causing impairment of cognitive functions including spatial and long-term memories in Y-maze and Morris water-maze tests. The primary cultured hippocampal neurons from KO mice had less branches and spines compared to the wild type. This morphological impairment was rescued by overexpression of DGKβ. In addition, overexpression of DGKβ in SH-SY5Y cells or primary cultured mouse hippocampal neurons resulted in branch- and spine-formation, while a splice variant form of DGKβ, which has kinase activity but loses membrane localization, did not induce branches and spines. In the cells overexpressing DGKβ but not the splice variant form, DGK product, PA, was increased and the substrate, DG, was decreased on the plasma membrane. Importantly, lower spine density and abnormality of PA and DG contents in the CA1 region of the KO mice were confirmed.These results demonstrate that membrane-localized DGKβ regulates spine formation by regulation of lipids, contributing to the maintenance of neural networks in synaptic transmission of cognitive processes including memory. DG regulates the functions of several enzymes including protein kinase C (PKC), Ras guanyl nucleotide-releasing protein (RasGRP), chimerins, and Unc-13. Then, the generated DG is phosphorylated by DG kinase (DGK) to produce phosphatidic acid (PA). PA is also important lipid second messenger regulating several enzymes including the mammalian target of rapamycin (mTOR) and atypical type of PKC. Therefore, DGK is thought to be a key enzyme regulating numerous cellular responses Many growth factors, neurotransmitters, and other extracellular signals evoke a rapid, but transient rise in the amounts of diacylglycerol (DG) and inositol 1,4,5-trisphosphate  are characterized by calcium-sensing regions, the recoverin homology (RVH) domain, and EF-hand motifs, in addition to two cysteine-rich regions, C1A and C1B, homologous to PKC C1 domain. Among Type I DGKs, the βTherefore, we investigated significance of DGKβ and its molecular mechanisms for neuronal functions using DGKβ KO mice which we developed, and show that spatio-temporal regulation of DGKβ results in proper spine formation contributing to long-term potentiation (LTP) and cognitive function including spatial and long-term memory.nd and 23rd exons among 25 exons coding DGKβ, suggesting the possibility that a part of N-terminal region of DGKβ was still expressed. Therefore, we determined its expression at protein level with Western blot analysis and immunohistochemistry using the antibody specific for N-terminus of DGKβ We produced DGKβ knock-out (KO) mice using the Sleeping Beauty transposon system t-test]. The following data analysis is less affected by swimming ability such as swim speed and locomotor activity The KO mice were viable and fertile with no significant difference in weight. We subjected DGKβ KO mice to several learning and memory tests to determine the involvement of DGKβ in cognitive function, because mouse DGKβ was detected in the hippocampus by immunohistochemistry , as repoNext, we tested whether hippocampal LTP in the CA1 region is impaired in DGKβ KO mice since it is essential for hippocampus-dependent contextual and spatial reference memory. In control slices from WT mice, high-frequency stimulation  of the Schaffer collateral/commissural pathways induced LTP in the hippocampal CA1 region, which lasted over 60 min  . As expeTo explore the molecular mechanisms underlying impairment of memory and LTP, we compared morphological difference between the primary cultured hippocampal neurons from DGKβ KO mice and their litter mate WT mice. There was no significant difference in the number of primary neurites from a single cell body of the primary cultured hippocampal neuron from WT and KO mice ; those oTo further investigate molecular mechanisms of neuronal morphological change by DGKβ, we compared the effect of overexpression of GFP-tagged DGKβ and its splice variant type which lacks 32 amino acids from C-terminus (defined as C-cut mutant) on the morphology of neurons and lipids contents. In the neuroblastoma cell line, SH-SY5Y, DGKβwas mainly expressed on the plasma membrane , consist2 in WT, while it was just 2.7±1.3 per 14 µm2 in KO mice  and Invitrogen , respectively. FuGene 6 transfection reagent was obtained from Roche Molecular Biochemicals . Adenovirus encoding NSE-tTA and TetOp-GFP used were described previously Pregnant mice (day 14) (C57BL/6JJmsSlc) for the primary culture were obtained from Japan SLC Inc. . DGKβ KO mice were produced using the Sleeping Beauty transposon system This study was approved by the Animal Experiment Committee of Gifu Pharmaceutical University , the Institutional Animal Care and Use committee of Kobe University  and the committee for Safe Handling of Living Modified organisms in Kobe University (H19-2). All procedures relating to animal care and treatment conformed to animal care guidelines of these committees.12. Genotyping of the mutant mice was conducted by PCR using the following primers: 5′-GAACAGAACAACAATAGCTTATGTTC-3′ and 5′-CTTGTGTCATGCACAAAGTAGATGTCC-3′ for the mutant allele, and 5′-GAACAGAACAACAATAGCTTATGTTC-3′ and 5′-TAAGTGGATATTAGCCCAGAAACTTAG-3′ for the WT allele. 455-bp and 540-bp bands are expected from the mutant and WT alleles, respectively. PCR conditions were as follows: 25 µl PCR volume, 1 cycle at 95°C for 15 min; 35 cycles at 95°C for 1 min, 55°C for 1 min, 72°C for 1 min; and 1 cycle at 72°C for 7 min. DGKβ−/− mice used for these studies were backcrossed to C57BL/6 mice for more than 9 generations.Dgkβ-mutant mice were generated using the Sleeping Beauty transposon system as described previously5′-AAATCCCGAATCTCTATCGTGCGGTGGTTG-3′ and 5′-GAAAGAAAGCCTGACTGGCGGTTAAATTGC-3′. To verify the absence of the vector concatemer at the donor site, genomic DNAs were digested with SpeI, fractionated on 0.8% agarose gel, and hybridized with the 0.8-kb neo probe, which is located in the vector backbone of the transposon vector. The neo probe was prepared by PCR-amplification from plasmid DNA using the following primers: 5′-TGGGATCGGCCATTGAACAAGATGGATTGC-3′ and 5′-CTCGTCAAGAAGGCGATAGAAGGCGATGCG-3′. Genomic DNA of a mouse cell line containing a single copy of the neo gene was used as a positive control to examine the sensitivity of the detection.Genomic DNAs from mouse tails were digested with BglII and NcoI, fractionated on 1% agarose gel, and hybridized with the 1.4-kb lacZ probe, which is located inside the transposon vector. The lacZ probe was prepared by PCR-amplification from a plasmid DNA using following primers: 5′-TGTGGACCCTTGAAGGACCATATTTTG and Reverse one wasCGGTTTCTTGTTCTTTTGATGAGGGAGCAG.-3′Total cellular RNA was extracted from forebrain of WT and KO mice using commercial reagents  according to manufacturer's instructions, and quantitated spectrophotometrically. Total RNA (0.2 µg) was reverse-transcribed into cDNA using the Super Script III First Strand Synthesis System for RT-PCR (Invitrogen) according to manufacturer's instructions. The cDNA was used as a template for following PCR to obtain quantitive value of DGKβ mRNA. Primers were synthesized commercially and the sequences were as follows. Forward'- primer was Various ages of brain were dissected from developing mice and homogenized using a Polytron  in ice-cold homogenate buffer . A portion was taken for determination of protein concentration and the remainder was mixed with 3×SDS sample buffer. Equal amounts of brain samples were subjected to 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli and the separated proteins were electorophoretically transferred to a polyvinylidine difluoride (PVDF) filter . The proteins separated by SDS-PAGE were transferred to a PVDF membrane and blocked with 5% skim milk in 0.01 M PBS containing 0.03% triton-X 100 (PBS-T). The membrane was immunostained with appropriate antibodies for 1 h at room temperature. After three rinses with PBS-T, the membrane was incubated with peroxidase-labeled anti-Rabbit IgG (Jacksons) or anti-mouse IgG (Jacksons) for 1 h at room temperature. After extensive washing with PBS-T, the immunoreactive bands were visualized using an enhanced chemiluminescence detection kit .2O2 for 20 min to inactivate endogenous peroxidase activity, with 5% NGS for 20 min to block non-specific binding sites and then with 0.1% phenylhydrazine to inactivate endogenous peroxidase activity for 20 min. The sections were then incubated with the antisera against DGKβ (1∶2000) in PBS-T for 72 h at 4°C. After washing with PBS-T, the sections were incubated for an additional 2 h with biotinylated goat anti-mouse IgG (1∶1000) , and then for 1.5 h with avidin-biotin-peroxidase complex (1∶1000) (Vector). After rinsing three times, the reaction product was visualized with 0.01% 3,3 diaminobenzidine , and 1% nickel ammonium sulfate in 0.05 M Tris-HCl (pH 7.6) with 0.0003% H2O2. Finally, the stained sections were mounted on gelatin-coated glass slides, dehydrated by a graded series of ethanol and covered with Entellan  for observation, and then photographed under a light microscope .DGKβ KO and WT mice were deeply anesthetized by the intraperitoneal injection of Nembutal (50 mg/kg). The mice were perfused with 0.9% NaCl through the left ventricle at a flow rate of 5–50 ml/min and then perfused with 250 ml of 0.1 M phosphate buffer  containing 4% PFA and 0.2% picric acid at 4°C. The brain was removed and immersed for 48 h in the same fixative. After washing with several changes of PB containing 30% sucrose for at least 2 days at 4°C, serial coronal sections of 20 µm in thickness were cut on a cryostat. These sections were immersed directly in PBS-T (0.1–0.3% Triton X-100) for at least 4 days at 4°C before use. The following steps were carried out at 25°C, unless otherwise specified. The frontal sections were washed for 5 min with PBS-T between each step. The frontal sections were preincubated with 0.3% HMice were anesthetized with Nembutal (50 mg/kg) and perfused with PBS (pH 7.4) until the outflow became clear, followed by 0.1 M PBS (pH 7.4) containing 4% PFA  for 15 min. Brains were removed and postfixed in the same fixative for 24 h at 4°C. Brain sections were equilibrated in 25% sucrose and quickly frozen in Tissue-Tek O.C.T. . After fixation with 0.2% glutaraldehyde and 1% formalin , tissues were stained with 5-bromo-4-chloro-3-indoly β-galactoside (Active motif) at 37°C for 24 h.5′-ATAGGATCCATGACAAACCAGGAAAAATGG-3′ and 5′-ATAGGATCCTCATTCCTTGCTTCGGTTT-3′. For making the C-cut mutant, the same sense primer and anti-sense primer, 5′-TTGGATCCTATTGTGCATGGGGTCTG-3′, were used. The PCR products were first subcloned into pCR™ 2.1  and sequenced. After digestion with Bam HI, the cDNA fragment was subcloned into the Bam HI sites of the pEGFP-C1 vector  (designated as BS931).The cDNA encoding DGKβ was produced by a polymerase chain reaction (PCR) with cDNA for DGKβ as the template. The sense and anti-sense primers used were The cDNA encoding GFP-DGKβ was cut out from BS931 by Nhe I/Mlu I digestion, followed by blunting using a blunting kit . After adding a Hind III/Sma I adaptor, the cDNA fragment was subcloned into the Hind III site of a pShuttle vector (BSAd 103). After linearization by Pme I, the pShuttle vector with the cDNA encoding fusion protein of GFP and DGKβ was co-electroporated with the pAdEasy backbone vector into BJ5183 bacterial cells. The recombination was checked by Pac I cut and the plasmids were purified by CsCl-banding. Approximately 10 µg of the purified plasmids digested by Pac I were lipofected into 50–70% confluent HEK293 cells plated on a 6-cm dish using FuGene 6 (Roche). The cells were scraped off at 7 days post-transfection and re-suspended in 1 ml of PBS(−). After sonication, 50–70% confluent HEK293 cells in a T75 flask were infected using the supernatant and cultured in DMEM supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), and 10% horse serum (Gibco BRL). To amplify further, the infection of cells using 30–50% of the viral supernatant and 50–70% confluent HEK293 cells in T75 flasks was repeated. Finally, the adenovirus was purified by CsCl-banding and titrated.2. The fetal bovine serum used was not heat inactivated.SH-SY5Y cells were cultured in DMEM/F-12 medium supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 µg/ml) . All cells were cultured at 37°C in a humidified atmosphere containing 5% CO5) were plated onto a glass-bottom culture dish  and then transfected with 2 µg of plasmid encoding GFP or GFP-DGKβ on the following day by lipofection using FuGene 6 according to the manufacturer's protocol. After culturing for 48 h, the cells were fixed with 4% paraformaldehyde (PFA) and 0.2% picric acid for 1 h at 4°C, and observed using confocal microscopy.SH-SY5Y cells  at pH 7.45 (Invitrogen). Primary culturing of hippocampal neurons was carried out using Nerve Cell Culture System . Briefly, hippocampal neurons were dissociated using dissociation solution and cultured using the glial-conditioned medium, in a glass-bottomed culture dish . Half of the medium was exchanged every 3 days with fresh medium.Fetuses were removed on embryonic days 17–18 from mice anesthetized by intraperitoneal injection of Nembutal . Hippocampi were dissected and placed in CaAfter culturing for 3, 10, or 15 days, adenoviruses NSE-tTA, TetOp-GFP, or TetOP-GFP-DGKβ were applied to a dish culturing hippocampal neurons. After 1 h incubation, the medium was washed well and cultured for a further 48 h. After fixation with 4% PFA and 0.2% picric acid at 4°C and washing with PBS-T, the fluorescence of GFP was monitored under confocal microscopy.The hippocampal neurons dissected form DGKβ KO mice or their litter mates WT mice were cultured for 3, 10, or 15 days, and fixed with 4% PFA and 0.2% picric acid at 4°C. After washing with PBS containing 0.03% triton X-100 (PBS-T), the cells were tritonized with 0.3% triton X in PBS(−) for 20 min and then blocked with 5% normal goat serum (NGS) for 1 h. The neurons were incubated with anti-MAP-2 antibody (1∶2000) for 16 h at 4°C. After rinsing three times, the neurons were visualized with Alexa 488-labeled goat anti-mouse IgG (1∶1000), followed observation under confocal microscope.The fluorescence of the GFP or Alxa 488 was observed with a confocal laser scanning fluorescent microscope  at 488-nm argon excitation using a 515–535-nm band pass barrier filter. The images were recorded as TIFF files. To count the number of neurites, branches, and spines, the image was analyzed with Neurolucida and Nurolucida Explorer software .Spontaneous alternation behavior in a Y-maze was assessed as a spatial working memory task. The Y-maze apparatus consisted of three identical arms (length 40× width 10× height 12 cm). Each mouse was placed at the end of one fixed arm and allowed to move freely through the maze during an 8-min session. The sequence of arm entries was recorded manually. An alternation was defined as entering each of the three arms consecutively. The maximum number of alternations was thus the total number of arms entered minus two, and the percentage of alternations was calculated as  ×100. The total number of arms entered during the session was also recorded.A circular pool (diameter 120× height 45 cm) was filled to a depth of 30 cm with water (21–23°C). Four equally spaced points around the edge of the pool were designated as four starting positions. A hidden platform (diameter 10 cm) was set 0.5 cm below the surface of the water in a fixed position. Mice were placed in the water facing the wall and trained with 4 trials per day for 5 days. In each trial, the starting position was changed, and the mouse swam until it found the platform, or after 60 s was guided to the platform; the mouse was then placed on the platform for 15 s before being picked up. Twenty-four hours after the last training trial the mice were given a prove test without the platform. In this test, each mouse was placed in the pool once and allowed to search for 60 s. Mean distance from the original location of the platform and the time spent in the quadrant where the platform had been, was recorded using a video camera-based Ethovision XT system .2, 5% CO2) artificial cerebrospinal fluid (ACSF) containing 126 mM NaCl, 5 mM KCl, 26 mM NaHCO3, 1.3 mM MgSO4• 7H2O, 1.26 mM KH2PO4, 2.4 mM CaCl2• 2H2O, and 1.8% glucose at room temperature (28°C). After a 2-h recovery period, a slice was transferred to an interface recording chamber and perfused at a flow rate of 2 ml/min with ACSF warmed to 34°C. Field excitatory postsynaptic potentials (fEPSPs) were evoked by a 0.05 Hz test stimulus through a bipolar stimulating electrode placed on the Schaffer collateral/commissural pathway and recorded from the stratum radiatum of CA1 using a glass electrode filled with 3 M NaCl. A single-electrode amplifier  was used to record the responses, and the maximal value of the initial fEPSPs slope was collected and averaged every 1 min (3 traces) using an A/D converter  and a personal computer. After a stable baseline was obtained, high frequency stimulation (HFS) of 100 Hz with a 1-s duration was applied twice with a 10-s interval and test stimuli were continued for the indicated periods.Preparation of hippocampal slices was performed as described previously The hippocampal region was cut out from the brains fixed as described above, and further immersed in 30% sucrose for 2–3 days. The tissue block was placed in 2% potassium dichromate for 2 days at 4°C and then in 2% silver nitrate solution for 2 days at 4°C in the dark. The block was cut into 60 µm thick sliced into distilled water. Finally, the sections were mounted onto slides, dried for 10 minutes, and dehydrated through 95% alcohol, 100% alcohol, clear in xylene.The anesthetized DGKβ KO and WT mice were perfused with 0.9% NaCl through the left ventricle at a flow rate of 5–50 ml/min and then perfused with 250 ml of 0.1 M phosphate buffer  containing 2% PFA and 2% glutaraldehyde at 4°C. The dissected brain was sliced with into sections of 1 mm thickness and 4 mm square, and immersed in the same fixative. The slice was further fixed in the 2% osmium tetroxide in 0.1 M PB, dehydrated through a graded series of ethanol and embedded in Epon (quetol-812). The ultrathin sections were mounted on the mesh and stained with 2% uranyl acetate and Reynolds solution, then observed and photographed with a JEOL JEM1200EX electron microscope.5 SH-SY5Y cells were lipofected with GFP-DGKβ, GFP-C-cut or GFP and cultured for 2 days. The cells were pre-incubated in phosphate-free medium for 30 min and then incubated with [32P]-monosodium phosphate (100 µCi/ml) for additional 2 h. Thereafter, the cells were harvested and lysed in 20 mM Tris-HCl (pH 7.5) containing 1 mM MgCl2, 1 mM EGTA, 1 mM PMSF, 20 µg/ml leupeptin. After sonication and centrifugation at 800×g, lipids including PA were extracted from the pellet, and separated by TLC. Spot of [32P]-PA was measured by BAS2500 . To measure membrane DG, membrane lipids were extracted from the SH-SY5Y cells normally cultured. The amount of DG was determined by its conversion into [32P]-PA by Escherichia coli DGK in the presence of [γ-32P ]-ATP. DGK assay was performed described as previously We measured membrane PA based on the methods by Aragones et al Escherichia coli DGK as described above. The half was subjected for measurement of PA using enzymatic reaction of lipase as described previously To measure PA and DG in the brain from WT and KO mice, mice were anesthetized by the intraperitoneal injection of Nembutal and perfused with 0.1 M phosphate buffered saline. The brain was removed and a section (5 mm ×2 mm ×1 mm thick) was cut out from the hippocampus and cerebellum region of WT and KO mice. The sections were sonicated  in 100 µl of PBS (-). Protein concentration of the homogenate was measured by Bradford methods. DG and PA were extracted from the homogenates using chloroform and methanol as described previously Figure S1Co-localization of endogenous DGKβ with PSD-95. The rat hippocampal primary neurons cultured for 21 days were fixed. Endogenous DGKβ was visualized with DGKβ antibody (1∶1000) followed by Alexa 594 conjugated anti-mouse IgG (1∶500), while PSD-95 was detected by rabbit anti PSD-95 antibody (Invitrogen) followed by Alxa 594 conjugated anti-mouse IgG (1∶500). Arrows indicate the spots where DGKβ colocalizes with PSD-95.(9.42 MB TIF)Click here for additional data file.Figure S2Temporally different effect of DGKβ overexpression on induction of primary neurite and neuronal branching in the primary cultured hippocampal neurons from WT mice. The mouse hippocampal neurons cultured for 3, 10, 15 days were infected with respective viruses. After 48 h of infection, the cells were observed under confocal microscopy and analyzed using Neurolucida software . Number of primary neurites (A) and branches per single neurite (B) in primary cultured hippocampal neurons overexpressing GFP-DGKβ (closed square + solid line) or control GFP (open circle + dotted line) were compared. day 5; n = 13 for GFP, n = 8 for DGKβ, day 12; n = 18 for GFP, n = 15 for DGKβ, day 17; n = 8 for GFP, n = 12 for DGKβ. * and ** represent P<0.05 and P<0.01, respectively.(9.04 MB TIF)Click here for additional data file.Figure S3The ontogeny and localization of DGKβ in mouse brain assessed by immunoblot analysis (A) and immunohistochemistry (B). (A) Brain homogenates (50 µg protein) from mice of various ages were fractionated by SDS-PAGE, transferred to a PVDF membrane, and incubated with the specific antisera against the DGKβ and β-actin to show that equal amounts of protein were applied in each lane. The intensity of each sample was analyzed by NIH Image software and results are expressed as a percentage of the intensity at P21. P means postnatal. Each bar represents the mean of 3 separate samples. The typical immunoblot of DGKβ and β-actin is shown in the panels below the bar graph. (B) Immunohistochemistry showing DGKβ localization in frontal sections of mouse brain. ctx, cerebral cortex; hip, hippocampus; cp, caudate putamen.(7.45 MB TIF)Click here for additional data file.Figure S4Inhibitory effect of C-cut mutant on the neurite induction. GFP-C-cut or GFP was expressed in the mouse cortex primary neurons cultured for 8 days. Forty eight hours later, the cells were fixed and observed under confocal microscopy. Red arrow indicates that the neuron expressing GFP-C-cut did not have neurites, while black arrows show that the neurons expressing no GFP-C-cut have neurites (Upper images). Similarly, the neurons expressing GFP alone have neurites (lower images). Bars are 10 µm.(7.07 MB TIF)Click here for additional data file.


Microsatellites in cDNA are useful as molecular markers because they represent transcribed genes and can be used as anchor markers for linkage and comparative mapping, as well as for studying genome evolution. Microsatellites in cDNA can be detected in existing ESTs by data mining. However, in most fish species, no ESTs are available or the number of ESTs is limited, although fishes represent half of the vertebrates on the earth. We developed a simple and efficient method for isolation of microsatellites from cDNA in fish.The method included normalization of 150 ng cDNA using 0.5 U duplex-specific nuclease (DSN) at 65°C for 30 min, enrichment of microsatellites using biotinylated oligonucleotides and magnetic field, and directional cloning of cDNA into a vector. We tested this method to enrich CA- and GA-microsatellites from cDNA of Asian seabass, and demonstrated that enrichment of microsatellites from normalized cDNA could increased the efficiency of microsatellite isolation over 30 times as compared to direct sequencing of clones from cDNA libraries. One hundred and thirty-nine (36.2%) out of 384 clones from normalized cDNA contained microsatellites. Unique microsatellite sequences accounted for 23.6% (91/384) of sequenced clones. Sixty microsatellites isolated from cDNA were characterized, and 41 were polymorphic. The average allele number of the 41 microsatellites was 4.85 ± 0.54, while the expected heterozygosity was 0.56 ± 0.03. All the isolated microsatellites inherited in a Mendelian pattern.Normalization of cDNA substantially increased the efficiency of enrichment of microsatellites from cDNA. The described method for isolation of microsatellites from cDNA has the potential to be applied to a wide range of fish species. The microsatellites isolated from cDNA could be useful for linkage and comparative mapping, as well as for studying genome evolution. Microsatellites are short segments of DNA in which a specific motif of 1–6 bases is repeated . Due to Lates calcarifer). From the 4800 ESTs, a total of 70 unique sequences containing microsatellites  from 130 clones were identified. Among the 70 microsatellites, 42 were CA-repeats, 23 GA-repeats, two GGA-repeats and three other types of repeats. These data indicate that unique microsatellite sequences accounted for 1.45% (70/4800) of cDNA clones in Asian seabass. CA- and GA-microsatellites were most abundant in cDNA of Asian seabass. However, they represent only 0.83% (40/4800) and 0.48% (23/4800) of cDNA clones from normalized cDNA libraries. Hence, straightforward random sequencing of clones from normalized cDNA libraries is not efficient for discovering microsatellites.In a previous study , we sequ10 and (GA)10 oligonucleotides, since these two types of microsatellites are most abundant in cDNA of Asian seabass. Two cDNA libraries were constructed, one enriched for CA-microsatellites and another for GA-microsatellites. From each library, 192 randomly picked clones were sequenced in both directions. Among the 192 clones from the cDNA library enriched for CA-repeats, 80 clones contained microsatellites. Of the 80 clones containing microsatellites, only 11 were singletons, and the remaining 69 were included in 8 clusters. A total of 19 (9.9%) unique microsatellites were obtained from the 192 sequences clones In order to increase the efficiency of enrichment of microsatellites, we tried to reduce the redundancy of cDNA by normalizing cDNA using duplex-specific nuclease (DSN)  before eP > 0.05). Therefore, microsatellites isolated from cDNA using the described method could be useful for linkage mapping and comparative mapping and studies on genome evolution.Sixty of 91 microsatellites isolated from the libraries enriched for CA- and GA-microsatellites had enough flanking regions for designing primers, and were characterized in a panel of 24 individuals previously used for characterization of microsatellites isolated from genomic DNA . Forty-oWe have developed a very simple and highly efficient method for identifying microsatellites from cDNA. Microsatellites isolated from cDNA showed polymorphism and a Mendelian pattern of inheritance. Therefore, the method will be ideal for isolation of microsatellites from cDNA of fish species where there are no EST sequences available or the number of ESTs is limited.In a previous study we sequenced 4800 ESTs from six normalized cDNA libraries of Asia seabass . MicrosaTotal RNA was isolated from brain of a 3-months old Asian seabass using Trizol (Invitrogen) according to the manufacturer's protocol. DNA residue in the RNA was removed with the treatment of DNAse (NEB). First strand DNA was synthesized using CDS primer [AAGCAGTGTATCAACGCAGAGTA(T35)], Smart oligo II A primer (AAGCAGTGTATCAACGCAGAGTACrGrGrG) and PowerScript reverse transcriptase (BD Bioscience) according to BD Bioscience's protocol. The reaction consisted of 3 μg total RNA, 1 mM CDS primer, and 1 mM smart oligo II A primer, 1 × first strand buffer (BD Bioscience), 2 mM DTT and 1 mM of each dNTP in 10 μl. The reaction was incubated at 42°C for 2 hours on a PTC-100 PCR machine (MJ research) and then cooled on ice.Second strand cDNA was synthesized using SMART cDNA technology. Briefly, the synthesized first strand cDNA was 1: 6 diluted in 1 × TE (pH 8.0), heated at 72°C for 10 min, and used in the synthesis of the second strand cDNA according to BD Bioscience's protocol. The 50 μl reaction comprised of 1 μl diluted first strand cDNA, 1 × buffer (BD Bioscience), five units of polymerase mix (BD Bioscience), 200 μM dNTPs, 0.25 μM smart PCR primer (AAGCAGTGTATCAACGCAGAGT). PCR was performed on PTC-100 PCR machine using the following program: 20 cycles of 95°C for 8 s, 68°C for 20 s and 72°C for 3 min.2 and 10 mM DTT] and 0.5 μl (1 U/μl) DSN (Evrogen). The 10 μl reaction was incubated at 65°C for 30 min on a PTC-100 PCR machine followed by heating at 95°C for 8 min to inactivate the DSN. The normalized cDNA was diluted 4 times with water, and amplified with the Smart PCR primer for 20 cycles as described in the above section.Normalization of cDNA was conducted using DSN (duplex-specific nuclease) (Evrogen)  accordinSal I and Not I respectively by the following 50 μl PCR reaction: 1 μl (20 times diluted) smart cDNA or normalized smart cDNA, 1 × advantage 2 buffer (BD Bioscience), 200 μM dNTPs, 1 μl (5 units) advantage 2 polymerase mix (BD Bioscience), 0.15 μM NotI-T25 primer [AATGTCGAGVGGCCGCGTAC(T)25], 0.15 μM SalI primer (TTGTAGCGTCGACTCACTATC), 0.015 μM SalI smart primer (TTGTAGCGTCGACTCACTATCAAGCAGTGTATCAACGCAGA). The PCR was performed on a PTC-100 PCR machine with the following program: 20 cycles of 95°C for 8 s, 68°C for 20 s and 72°C for 3 min.To produce directional microsatellite-enriched cDNA libraries, the 5' and 3' ends of the normalized cDNA were annealed a linker with a cutting site 10 or (GA)10 in 65 μl 6 × SSC at 55°C for 25 min. DNA hybridization products (65 μl) were captured with 35 μl (ca. 350 μg) streptavidin coated beads (Pierce) (suspended in 6 × SSC) which were washed twice in 1 × TE (pH 8.0) and twice in 6 × SCC before capture at room temperature. Beads capturing microsatellite-enriched cDNA were washed twice in 2 × SSC containing 0.1% SDS and twice in 1 × SSC at room temperature, and then a final wash in 1 × SSC at 55°C for five min. The captured cDNA was eluted with 30 μl water and PCR-amplified in a reaction of 25 μl consisted of 3 μl eluted cDNA, 200 nM SalI primer, 200 nM NotI-T25 primer, 200 μM dNTPs, 1 × PCR buffer, and two units of polymerase mix (BD Bioscience). The PCR was carried out on a PTC-100 PCR machine using the following program: 30 cycles of 95°C for 8 s, 65°C for 20 s and 72°C for 3 min. PCR products were cleaned and concentrated using glassmilk (Gen 101).Microsatellites in cDNA were enriched by using biotinylated oligonucleotides and streptavidin-coated magnetic beads. Briefly, 1 μg cDNA in 6 × SSC was denatured at 98°C for 5 min, followed by hybridization with 1 μl 10 pmol/μl biotinylated (CA)alI and NotI using the following protocol: 20 μl (ca. 500 ng) cleaned cDNA, 3 μl 10 × SalI buffer, 1 μl (20 units) SalI (NEB) and 1 μl (10 units) NotI (NEB) at 37°C for 2 hours. After digestion, the cDNA was electrophoresed on 1% low melt gel (BIO-RAD). Fragments between 500 bp and 1200 bp were excised and cleaned using glassmilk (Gen 101). Approximately 50 ng cDNA was ligated to 25 ng pCMV-SPORT-6 vector (Invitrogen) which was used to transform XL-blue supercompetent cells (Stratagene). Schematic presentation of the method for microsatellite enrichment from normalized cDNA is shown in Figure The microsatellite-enriched cDNA PCR products was digested with Sst Base). DNA sequences of the polymophic microsatellites were deposited in GenBank with the accession numbers: EF210110–EF210125 and FJ535708–FJ535732.White colonies were picked and arrayed into 96-well plates containing 40 μl LB liquid medium with 100 μg/ml ampicillin in each well. The 96 well plates were cultured at 37°C for 16–18 hours without shaking. Inserts of each colony were PCR amplified using two microliter cell culture in LB as template, low concentration (50 nM) of M13PUC forward (5' CCCAGTCACGACGTTGTAAAACG 3') and reverse primers (5' AGCGGATAA-CAATTTCACACAGG 3') with the following PCR program: 94°C for 5 min followed by 35 cycles of 94°C at 30 s, 55°C for 30 s and 72°C for 48 s, with a final extension at 72°C for 5 min. Two microliters of colony PCR products were directly sequenced in both directions using M13PUC-F/M13PUC-R primers and BigDye kit on an ABI3730xl sequencer (both from Applied Biosystems). Forward and reverse sequences were assembled using software Sequencher (GeneCodes). Primers were designed for a subset of microsatellites in the flanking regions using PrimerSelect (Dnastar). One primers of each pair was labeled with a fluorescent dye Hex or Fam . Cycling conditions were: 94°C for 2 min followed by 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 30 s, with a final extension at 72°C for 5 min. Fluorescence-based genotyping of 24 unrelated Asian seabass individuals originated from Southeast Asia and Australia was conducted using an automated DNA sequencer ABI 3730xl (Applied Biosystems). Each microsatellite was examined for genotyping errors using MicroChecker [PCR amplification of microsatellites was performed on a PTC-100 thermal cycler in a 25 μl reaction volume containing 100 ng DNA, 1 × PCR buffer [50 mM KCl, 10 mM Tris-HCl (pH 8.8), 1.5 mM MgClcDNA: complementary DNA; EST: expressed sequence tag; DSN: duplex-specific nuclease.GHY planned and started the project, drafted and determined the final version of the manuscript. ZZY, WCM and XJH conducted the experiments. All authors have read and approved the final version of the manuscript.