Dmrt1 suggested that SDGs evolve from downstream genes by gene duplication. Orthologous sequences of the major genes of the mammalian sex determination pathway have been reported in the rainbow trout but the map position for the majority of these genes has not been assigned.Rainbow trout have an XX/XY genetic mechanism of sex determination where males are the heterogametic sex. The homology of the sex-determining gene (SDG) in medaka to Amh, Dax1, Dmrt1 and Sox6) were tested for linkage to the Y chromosome of rainbow trout. We exclude the role of all these loci as candidates for the primary SDG in this species. Sox6i and Sox6ii, duplicated copies of Sox6, mapped to homeologous linkage groups 10 and 18 respectively. Genotyping fishes of the OSU × Arlee mapping family for Sox6i and Sox6ii alleles indicated that Sox6i locus might be deleted in the Arlee lineage.Five loci of four candidate genes (Sox6 loci supports previously suggested homeology between linkage groups 10 and 18. Enrichment of the rainbow trout genomic map with known gene markers allows map comparisons with other salmonids. Mapping of candidate sex-determining loci is important for analyses of potential autosomal modifiers of sex-determination in rainbow trout.Additional candidate genes should be tested for their linkage to the Y chromosome. Mapping data of duplicated  Dmy, is a homologue of a downstream gene of the pathway in mammalian species  and a 649 bp promoter sequence of Dmrt1 [Genbank: FJ617281] identified a number of transcription factor binding sites or cis regulatory elements  and fugu (Takifugu rubripes). PCR amplification of the fourth intron of Sox6 from OSU and HC produced two products of differing size in each of the clonal lines. Following cloning and sequencing of all four products, OSU products were determined to be 984 and 1067 bp in size, while the sizes of the recovered products in HC were 984 and 1097 bp  indicated that Sox6 is duplicated in the rainbow trout.Boundaries of eight putative introns were identified in Sox6 allele in Arlee. Sequence comparison revealed a polymorphism between OSU and Arlee parental lines that resulted in the loss of an AvaII restriction site in Sox6ii of OSU .PCR amplification of the same intron from Arlee produced only one product 984 bp in size. Cloning and sequencing of the product did not show any sequence variants supporting the presence of a second SU Table . PCR-RFLSox6i to linkage group 18 (acrocentric chromosome 26) and Sox6ii to linkage group 10 (long arm of chromosome 6) supporting their homeology  OSU – Sox6i/OSU – Sox6ii (Genotype 1 in figure Sox6i/A – Sox6ii (Genotype 2) (3) OSU – Sox6i/A – Sox6ii (Genotype 3) (4) A – Sox6i/OSU – Sox6ii. This last genotype was not observed in the O × A family (figure If we assume that Arlee (A) has two identical Genotype  (3) OSU Sox6 loci failed to amplify Sox6i allele in Arlee because of a mismatch in the primer sequence, or because of deletion of Sox6i in the Arlee lineage, Sox6i allele in Arlee can be genotyped as a null allele. Four genotype classes are expected in the O × A progeny if Sox6i is genotyped as a null allele: (1) OSU – Sox6i/OSU – Sox6ii (Genotype 1 in figure null/A – Sox6ii (Genotype 2) (3) OSU – Sox6i/A – Sox6ii (Genotype 3) (4) A -null/OSU – Sox6ii (Genotype 4). Genotype analysis of the O × A mapping family showed that 23 out of 73 doubled haploids were of genotype 1, 20 doubled haploids were of genotype 2, another 20 doubled haploids were of genotype 3 and only 9 were of genotype 4. Chi-square analysis did not show significant deviation of the observed proportions for the different genotype classes from the theoretical proportions expected under this model .If we assume that primer combination used to amplify different null/OSU – Sox6ii) genotype can also be explained by failure of the AvaII restriction assays. Genotyping was however confirmed by digestion with NlaIII which also produces a distinctive restriction profile for each of the four genotype classes of the mapping family and thus ruling out this possibility (data not shown).The  and three WT1 loci [We tested five loci for their linkage to the Y chromosome of rainbow trout in this study. In addition, three WT1 loci  have alsWT1 loci .Sox gene (Sry-related HMG box) family have been shown to be involved in the sex differentiation pathway or expressed in embryonic testes or ovaries [Dmrt1, orthologous sequences of Dmrt2 and Dmrt4 have been isolated from rainbow trout [Pax2a as a gene that is expressed early in gonadal development and displays a testis-specific expression profile. Our study identified potential binding sites of Pax2 in the promoter sequences of Dmrt1 and Dax1 suggesting that members of this gene family should be explored in future analyses. Although our analysis focused on candidate genes that already have sequences deposited in public databases, paralogous loci might still be isolated and would also be strong candidates for sex-determination based on the model presented for evolution of sex-determining systems [A number of candidate genes can still be tested for their linkage to the sex locus of rainbow trout. In mammalian species, ten out of the 30 genes discovered so far in the  ovaries ,25. Rain ovaries ,26. In aow trout . Studyinow trout  identifiIt is largely accepted that the sex-determining system in rainbow trout is genetic with male heterogamety. Nonetheless, Quillet et al  reportedSox6 locus. Inheritance pattern of Sox6 loci in O × A mapping family indicated possible deletion of Sox6i locus in the Arlee lineage. Our mapping data supports previously suggested homeology between linkage groups 10 and 18 in rainbow trout, and adds four known genes to its genomic map. Enrichment of the rainbow trout genomic map with type I markers allows the identification of conserved syntenic blocks between rainbow trout and other salmonid species, which would eventually allow for understanding the molecular organization of genomes following genome duplication. Although we did not find the primary sex-determining gene in this study, our data is of importance for analyses of potential autosomal modifiers of sex-determination in rainbow trout.Using linkage analysis, we excluded the role of five candidate loci as the primary sex-determining gene in rainbow trout. We also identified a duplication of the Two mapping families and their respective linkage maps have been used for this study. Mapping families are doubled haploid fish generated from F1 hybrids between two clonal lines using androgenesis. Clonal lines were produced by androgenesis and gynogenesis ,30. EachSox6, Dmrt1 and Dax1.This family was produced from the F1 hybrid of the Oregon State University  and Hot Creek  clonal lines. The family was previously described in Zimmerman et al  where itSox6 and Amh. Synteny between the O × H and O × A linkage maps has been established [This family was produced by androgenesis from a cross between the OSU (XX) and Arlee  clonal lines, previously used to generate a dense linkage map and is described in Young et al  and Nichablished .Sox6, Dmrt1 and Amh through comparisons of their cDNA sequences from Genbank  or TIGR gene index  with genomic sequences of Takifugu rubripes and Danio rerio. Primer sequences are listed in Table In order to amplify genomic DNA fragments of non-coding regions of candidate genes, primers were designed to anneal within exons flanking tentative introns. Boundaries between exons and introns were predicted for 2, 10 pmoles of each primer, 0.25 mM dNTPs  and 1.5 U of Taq DNA Polymerase .All PCR reactions were performed using a Thermolyne Amplitron II thermocycler. PCR reaction conditions began with denaturation at 94°C for 2 minutes, followed by 30 cycles of 94°C for 45 seconds, 60°C for 45 seconds and 72°C for 45 seconds. The reaction ended with a final extension step of 72°C for 2 minutes. All reactions were 20 μl in volume containing: 50 ng DNA template, 1× PCR buffer , 2.5 mM MgClFollowing the amplification of non-coding regions of candidate genes, PCR amplification products were resolved in a 2% agarose gel stained with ethidium bromide. Desired DNA fragments were gel-purified using Gene Clean , cloned into the pGEM-T Easy plasmid system  and then sequenced at the Washington State University Laboratory for Biotechnology and Bioanalysis. Target gene sequence verification was performed using the BLASTN and BLASTX algorithms of the NCBI website. Sequence polymorphisms between parental alleles were identified by aligning them using Sequencher 3.11 software . Because cloning can introduce artifact sequence differences, polymorphisms were confirmed by direct sequencing of genomic DNA amplification products using a nested primer (nested primer sequences are not shown).Dax1 and Dmrt1 from a λ Zap II  genomic library created from an O × H F1 hybrid. Following sequencing of the Dax1 and Dmrt1 lambda library clones by PCR walking, the structure of the Dax1 gene was deduced based on sequence alignment with the relevant sequences of Dicentrarchus labrax and Oreochromis niloticus. Intron and promoter sequences of Dmrt1 were identified based on alignment of the recovered genomic clone with rainbow trout Dmrt1 published cDNA sequence.A PCR-based approach  was used to isolate lambda clones of Sox6, Dmrt1 and Amh have sequence substitutions between alleles of the different parental lines that alter recognition sequences of restriction enzymes (RE). Utilizing these differences, doubled haploid fish of the mapping family were genotyped for the parental allele containing the polymorphic restriction site by PCR amplification, followed by RE digestion. Products of the RE-digest were size-fractionated in 1.5% agarose gels. Appropriate restriction enzyme digestions were done in 20 μl volume reactions according to the manufacturer's directions . Gene-specific primers utilized in RFLP-genotyping are listed in Table Non-coding sequences of the candidate genes Dax1. The polymorphism did not change any published recognition sequence of a RE. A Taqman assay  was used to score doubled haploids of the O × H mapping family for having either OSU or HC alleles. Primers and probe sequences used in the Taqman assay are listed in Table A single nucleotide polymorphism (SNP) was detected between OSU and HC in the deduced promoter region of Sox6 fourth intron was found to have a different size among the different Sox6 alleles . Genotyping data of candidate genes was incorporated into the pre-constructed O × H and O × A linkage maps using Map Maker 3.0/EXP . Marker Dax1 and 0.6 kb region upstream of Dmrt1 coding regions were sequenced from the isolated lambda clones. The promoter sequences were analyzed for the presence of putative regulatory elements for transcription factors known to be involved in sex differentiation using MatInspector software .A 2.2 Kb region upstream of MA drafted the manuscript, assisted in designing the study, recovered genomic sequences and genotyped doubled haploid fish of reference families. JB assisted in genotyping analyses and provided technical assistance. RD conducted linkage analysis on the reference families. GT conceived the study, participated in its design and coordinated the roles of the authors. All authors read and approved the final manuscript.


We identified avian influenza virus A (H5N1) infection in a child in Bangladesh in 2008 by routine influenza surveillance. The virus was of the same clade and phylogenetic subgroup as that circulating among poultry during the period. This case illustrates the value of routine surveillance for detection of novel influenza virus. Human infection with highly pathogenic avian influenza (HPAI) virus A (H5N1) has been associated with severe disease  and often with death  and the International Centre for Diarrhoeal Disease Research, Bangladesh  established nationwide hospital-based surveillance to identify clusters of persons with life-threatening influenza virus infections . His lungs sounded clear. The patient again visited the clinic on February 5 and February 10 and was afebrile on both occasions with a mildly elevated respiratory rate (38 breaths/min) and clear lungs. The mother reported that the child had had loose stools (possibly associated with amoxicillin) on February 10, but she denied that he had diarrhea. The child was never hospitalized; he completed a 14-day course of amoxicillin for suspected enteric fever and recovered fully. His blood culture was negative for any organism, and his final diagnosis was upper respiratory tract infection. On February 13, a convalescent-phase serum sample was collected as part of routine surveillance.Culture of the child’s nasopharyngeal wash sample showed cytopathic effects consistent with influenza virus infection; an aliquot reacted to influenza virus A antiserum but not to antiserum to subtypes H1 or H3. An aliquot of the viral culture material was shipped frozen on April 22, 2008, to CDC, where the isolate was identified as HPAI (H5N1) virus by real-time reverse transcription–PCR. The clinical specimen was recultured on embryonated chicken eggs. The complete genome was sequenced (GenBank accession nos. FJ573465–FJ573472). The virus was identified as an H5N1 clade 2.2 strain on the basis of the hemagglutinin sequence . After iWe report HPAI (H5N1) in a child in Bangladesh; the infection was confirmed by virus isolation from an upper respiratory specimen and by serologic testing. The source of the child’s subtype H5N1 infection is uncertain. One potential exposure was the healthy-appearing chicken that was brought inside the home. The child did not have direct contact with the chicken, although indirect contact was suggested because his mother handled him after slaughtering the chicken. The owner of the poultry shop where the chicken was purchased reported that 5%–10% of chickens had died each day during January 2008 (In Bangladesh and other countries with influenza (H5N1) outbreaks among poultry, surveillance for human subtype H5N1 cases is focused on hospital-based case finding for febrile patients with severe acute respiratory illness. This child was not suspected of having subtype H5N1 infection and had had no known poultry contact; his illness would not have met standard criteria for subtype H5N1 testing (The 1 in 5 sampling frame, a major limitation of this study, raises the possibility that undetected mild cases of subtype H5N1 infection have occurred in children in this population. Other limitations include the elapsed time between illness onset and investigation and the identification of only 1 case.The public health value of identifying the cause of severe acute respiratory illness clusters with pandemic potential is clear. This case highlights the value of routine outpatient surveillance for detecting both seasonal and novel influenza A viruses, particularly in settings in which subtype H5N1 strains circulate among poultry. Because exposure of subtype H5N1 to humans increases its opportunities for genetic mutation or reassortment, or both, with human influenza A viruses (


We also studied the role of those allele losses in gallbladder carcinoma pathogenesis by examining 45 microdissected normal and dysplastic gallbladder epithelia accompanying gallbladder carcinoma, using 17 microsatellite markers. Overall frequencies of loss of heterozygosity at 3p (100%), 8p (100%), 9q (88%), and 22q (92%) sites were very high in gallbladder carcinoma, and we identified 13 distinct regions undergoing frequent loss of heterozygosity in tumours. Allele losses were frequently detected in normal and dysplastic gallbladder epithelia. There was a progressive increase of the overall loss of heterozygosity frequency with increasing severity of histopathological changes. Allele losses were not random and followed a sequence. This study refines several distinct chromosome 3p, 8p, 9q and 22q regions undergoing frequent allele loss in gallbladder carcinoma that will aid in the positional identification of tumour suppressor genes involved in gallbladder carcinoma pathogenesis.Our recent genome-wide allelotyping analysis of gallbladder carcinoma identified 3p, 8p, 9q and 22q as chromosomal regions with frequent loss of heterozygosity. The present study was undertaken to more precisely identify the presence and location of regions of frequent allele loss involving those chromosomes in gallbladder carcinoma. Microdissected tissue from 24 gallbladder carcinoma were analysed for PCR-based loss of heterozygosity using 81 microsatellite markers spanning chromosome 3p  87, 432–440. doi:10.1038/sj.bjc.6600490www.bjcancer.comCancer Research UK© 2002  Gallbladder carcinoma (GBC) is a relatively uncommon neoplasm which demonstrates considerable geographic and gender variation in incidence . For unkIt is now well recognised that tumourigenesis is a multistep process resulting from the accumulation of sequential genetic alterations . In addiOur recent genome-wide allelotyping analysis on GBC indicated that allelic losses at multiple sites of the genome are frequent in this neoplasm . Six (25%) were well differentiated, eleven (46%) were moderately differentiated, and seven (29%) were poorly differentiated tubulo-papillary adenocarcinomas. The majority of the tumours were advanced GBCs  with invasion of the gallbladder serosa; the remaining  were early GBCs, with invasion of the submucosa  or muscularis propia  of the gallbladder.Forty-five histologically discrete foci of non-invasive gallbladder epithelia were identified adjacent to 20 GBCs, each consisting of at least 1000 cells. These included 17 histologically normal epithelia and 28 high-grade dysplasias , 8p (n=14), 9q (n=29) and 22q (n=12) regions showing high frequencies of LOH in the genome-wide allelotyping analysis on GBC  spanning a total of 12 chromosomal regions frequently deleted in invasive GBC , 8p n=1, 9q  to examine LOH at 12 chromosomal regions located on the four chromosomal arms examined. To determine whether the losses were progressive in the sequential development of GBC, we determined the frequency of loss of individual microsatellite markers (n=17) and chromosomal regions (n=12) using the Fractional Allele Loss (FAL) index and the Fractional Regional Loss (FRL) index, respectively. The FAL index is defined as the total number of microsatellite markers with LOH in an epithelial sample divided by the total informative markers in the corresponding normal DNA. The FRL index is defined as the total number of chromosomal regions with LOH divided by the total number of informative regions in the corresponding normal DNA.The data were analysed using a series of P<0.05 were regarded as statistically significant.Statistical analysis was performed using the nonparametric Wilcoxon and Fisher Exact tests. The cumulative binomial test was used to examine the likelihood that the occurrence of a particular event  occurs at a particular probability when observed in repeated trials. When the results are compared with a chance occurrence or nonoccurrence, the particular probability of comparison is 0.5. Probability values of Overall frequencies of allelic loss at any 3p , 8p , 9q , and 22q  sites were very high in GBCs. Most of the sites of allelic loss at all chromosomal arms examined were localised, and the extent of the partial allele losses was used to identify 13 discrete minimal regions of non-overlapping allele losses . The patterns of LOH and frequencies of allelic loss at different critical regions identified are shown in Table 2Although most of the losses at all chromosomal arms examined were localised, the data demonstrate that in chromosomes 3p, 8p and 9q the most frequently observed pattern was loss of two or more regions. Thus, allelic loss of a single region in each of those chromosomes by itself was a relatively infrequent event. Two or more regions were lost in 84% at 3p, 73% at 8p, 63% at 9q, and 52% at 22q. Cluster analysis to examine if allelic loss at one chromosomal region was linked to changes at another region  and dysplastic  foci examined . The FALThe pattern of allelic loss was not random, and losses at one or more 3p (54%) and 8p (50%) regions were the most frequently detected abnormalities in histologically normal epithelium . While 9Figure 6n=11) and dysplastic (n=19) epithelial specimens that were informative for at least one marker in each chromosomal arm examined. From these specimens only a single dysplastic focus showed no LOH at any chromosomal arm and was excluded from this analysis. Three patterns of allelic loss were discerned in the 30 histologically normal, dysplastic and neoplastic foci: (a) Early pattern, with only 8p loss or 8p accompanied with 3p. (b) Intermediate pattern, with only 9q loss or 9q accompanied with 3p and/or 8p. (c) Advanced pattern, with 22q loss accompanied with 8p+3p or 8p+9q or 8p+3p+9q. Of interest, normal gallbladder epithelium had only early (55%) and intermediate (45%) patterns, while tumours had advanced (71%) and intermediate (29%) patterns. Dysplastic gallbladder epithelia demonstrated the entire spectrum of patterns, including the advanced (56%), intermediate (30%) and early (14%).To determine the sequential molecular changes involved in the development of GBC, we analysed the pattern of allele losses detected in the GBC tumours and their accompanying normal and dysplastic epithelia. We considered only 14 tumours and 30 accompanying normal (P=5.3×10−22).Previous studies in gallbladder carcinoma and other neoplasms demonstrated that at any one locus, loss of parental alleles was not random, and that there was a strong tendency for the identical allele to be lost in all non-neoplastic and neoplastic foci examined . We refeP=9.1×10−26).Because in the GBC there is a close morphological relationship between invasive carcinoma and its dysplastic lesions  we then We detected a relatively high frequency of MSI at one or more 3p, 8p, 9q and 22q chromosomal loci in one of 13 (8%) of histologically normal foci, in four of 54 (7%) dysplastic foci and in six of 12 (50%) invasive carcinomas examined (data not shown).Our recent genome-wide allelotyping analysis indicated that allelic losses at multiple sites of the genome are frequent in GBC, and indicated for the first time that in addition to 3p and 8p, LOH on 9q and 22q may also play a role in the pathogenesis of this neoplasm , 8p (100%), 9q (88%), and 22q (92%) sites were very high in our tumours. Overall, thirteen distinct sites of frequent allele loss in GBC at chromosome 3p, 8p, 9q and 22q (summarised in RARβ) gene (FHIT) gene, spans the FRA3B fragile site at 3p14.2 (ROB01 (DUTT1), has been cloned residing in the U2020 3p12 deletion region at marker D3S1274  in our GBC cases. Recently, a new candidate TSG located in this region, the human RAS effector homologue (termed RASSF1A) gene, has been shown to have tumour suppressing function and undergoes epigenetic inactivation in several cancers  with frequent allelic loss in this tumour. Several candidate TSGs have been detected in those 3p regions with frequent allele losses in GBC. One candidate in the 3p22-24 region is the retinoic acid receptor-beta ( D3S1274 . One of (PRLTS) candidate TSG is centromeric to our 8p21 minimal region  gene, a candidate TSG at this region  occurring early during the sequential pathogenesis of GBC. The present findings of frequent chromosome 3p, 8p, 9q and 22q allele losses in non-malignant gallbladder epithelia confirm and greatly extend the findings that molecular changes commence early  during the sequential pathogenesis of GBC. Our major findings regarding the molecular pathogenesis of GBC are: (1) molecular changes preceded the onset of histologically recognisable changes and 88% of the normal histologically normal foci have allele loss at one or more chromosomal regions examined. (2) There was a progressive increase of the overall LOH frequency expressed by the FAL and FRL indices, with increasing severity of histopathological changes. The development of epithelial cancers requires multiple mutations , are present in many cancers, including GBC (In summary, allele losses of chromosome 3p, 8p, 9q and 22q regions are frequent in GBC. Our data identifies 13 distinct regions of loss on those chromosomal arms, many of which harbour one or more candidate TSGs, which may play a role in GBC pathogenesis. These regions have previously been reported to be frequently lost in several human cancers, suggesting that may harbour TSGs whose inactivation may be critical to the process of tumourigenesis. In addition, our findings in the non-malignant gallbladder epithelium indicate that multiple, non-random and sequential allele specific abnormalities commence early in the multistage pathogenesis of GBC. These findings should be useful for the identification of the TSGs involved in the pathogenesis of GBC, with the potential for defining molecular markers for early detection as well as for the development of gene therapy strategies of this highly malignant neoplasm.


Gad1, Gabrb3 or Viaat genes leads to the development of non-neural developmental defects such as cleft palate. Studies of the Gabrb3 and Gad1 mutant mice have suggested that GABA function could be required either in the central nervous system or in the palate itself for normal palatogenesis.Previous studies have shown that disruption of GABA signaling in mice via mutations in the Gad1 or Viaat function is required in the fetal CNS for normal palate development. We used oral explant cultures to demonstrate that the Gad1 and Viaat mutant palates were able to undergo palatogenesis in culture, suggesting that there is no defect in the palate tissue itself in these mice. In a second series of experiments we found that the GABAA receptor agonist muscimol could rescue the cleft palate phenotype in Gad1 and Viaat mutant embryos. This suggested that normal multimeric GABAA receptors in the CNS were necessary for normal palatogenesis. In addition, we showed that CNS-specific inactivation of Gad1 was sufficient to disrupt palate development.To further examine the role of GABA signaling in palatogenesis we used three independent experimental approaches to test whether Gad1 and Viaat in the central nervous system for normal development of the palate. We suggest that the alterations in GABA signaling lead to non-neural defects such as cleft palate as a secondary effect due to alterations in or elimination of fetal movements.Our results are consistent with a role for  Gad1, Gabrb3 or Viaat (Slc32a1) has been an enigma Gad1), GABA vesicular transport (Viaat) and the postsynaptic response to GABA (Gabrb3). The most prominent phenotype noted by us and other investigators has been the surprising presence of a cleft palate in neonatal mice homozygous for null mutations in Gad1, Viaat and Gabrb3GAD1 or GABRB3 have been reported Gad1, Viaat and Gabrb3 mice might lead to new insights into the origin of human oral clefts, a common congenital disorder.The mechanism leading to non-neural developmental defects in mice homozygous for null alleles of the genes Gad1 and Gabrb3 mutant mice. These studies have drawn different conclusions depending on the gene examined and the experimental approach taken. In the case of Gabrb3, two studies support a role in non-neural cell types for normal palatogenesis. In one case, transgenic expression of Gabrb3 from a neuron-specific enolase promoter failed to complement the cleft plate phenotype of a Gabrb3 null mutation, suggesting that non-neural expression of Gabrb3 was required for normal palate formation Gabrb3 gene, again suggesting that Gabrb3 function was required in the palate or other craniofacial structures for normal palatogenesis Gad1, previous studies indicated that the cleft palate phenotype of Gad1-/- pups was due to a lack of fetal oral movements as well as inhibition of palate shelf elevation due to the abnormal position of the tongue between the shelves Gad1 phenotype was consistent with a requirement for GABA signaling in the CNS for normal fetal movement that in turn allows normal palate development. However, several papers have reported GABA, Gabrb3 or Gad1 expression in developing craniofacial structures such as the palate, oral epithelium, tooth placodes or condensations, tooth buds and palate medial edge epithelium Gad1 mRNA or protein as well as GABA in these structures is consistent with a functional role for GABA signaling in the palate. In addition, Gad1 gene expression has been detected in several epithelial placodes as well as in non-neural tissues that are developmental signaling centers, suggesting a role for GABA in developmental processes outside of the CNS Viaat, cleft palate and body wall phenotypes were noted in a previous analysis of a Viaat knockout mouse Viaat mutants.Several studies have been performed to determine whether GABA signaling is required in the CNS or in peripheral tissues for normal palate formation in Gad1 and Viaat mutant mice are due to loss of gene function within the CNS during development. We performed a pharmacological rescue experiment to show that the GABAA agonist muscimol can suppress the cleft palate phenotype in Gad1 and Viaat mutant embryos. The ability of muscimol to rescue the cleft palate phenotype of Gad1 and Viaat mutants suggests that multimeric GABAA receptors are downstream of Gad1 and Viaat in palate formation, a pathway that is most consistent with GABA signaling in the CNS. In addition, we used a serum free explant culture system to show that oral explants derived from laczGad1 -/- and lacZViaat -/- embryos were competent to undergo normal palate shelf elevation and fusion when removed from their normal oral context. This experiment provided additional evidence that Gad1 and Viaat function are not directly required for palate formation. A third experimental approach was to inactivate Gad1 specifically in neural precursor cells throughout the CNS. We took advantage of a Gad1 conditional allele to demonstrate that an early neural precursor specific knockout of Gad1 was sufficient to cause a cleft palate phenotype in mutant embryos. Our work strongly supports the idea that the cleft palate phenotype in Gad1 and Viaat knockout mice is due to a loss of GABA signaling in the CNS rather than in craniofacial tissues. We interpret our results in the context of other studies that have documented a role for fetal movement in the development of tissues and structures outside of the CNS. We suggest that the phenotypes seen in the Viaat and Gad1 knockout mice belong to a family of fetal defects in non-neural tissues that are caused by a loss of normal fetal movements due to defects in CNS or muscle function during development.In this study we used three independent experimental approaches to show that the non-neural defects in lacZViaat knockin/knockout allele was generated by gene targeting in ES cells. Genomic clones containing the mouse Viaat locus were isolated from a 129/SvEv lambda phage library. A 5.2-kb fragment immediately upstream of the translation start site was used as a 5′ arm and a 2.8-kb fragment starting downstream of the start codon was used as a 3′ arm. A β-galactosidase reporter/neomycin resistance cassette was placed into an NcoI site in the Viaat first exon. This NcoI site includes the start codon of the Viaat gene Viaat knockout mice used in this study had been crossed for 4–10 generations to the C57Bl/6J background. The lacZGad1 knockin-knockout mice have been described previously Gad1 gene was obtained from Dr. Richard Palmiter. This strain has been described previously All work with mice conformed to the stipulations of the University of Georgia Institutional Animal Care and Use Committee. The University of Georgia animal welfare assurance number is A3437-01 which expires on 11/30/2011. The For paraffin sections, embryos were fixed in Bouin's solution at 4°C from 3 hours to overnight depending on the embryonic stage and washed in phosphate buffer (pH 7.3) overnight. Then the embryos were then dehydrated in a graded series of ethanol, equilibrated with xylene, embedded in paraffin and sectioned at a thickness of 10 µm. Sections were stained with Mayer's hematoxylin (Sigma) and eosin solution after dewaxing with xylene, and mounted on slides.Viaat embryos. The embryos were fixed in 0.4% paraformaldehyde (PFA), 100 mM sodium phosphate pH 7.3, 2 mM MgCl2, 5 mM EGTA for 30–60 minutes and then rinsed in 100 mM sodium phosphate pH 7.3, 2 mM, MgCl2, 0.01% sodium deoxycholate, 0.02% igepal for at least 3 hours and stained in 100 mM sodium phosphate pH 7.3, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% igepal, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 1 mg/ml X-gal at 37°C. After staining, embryos were washed in PBS and postfixed in 4% PFA/PBS for 1 hour at room temperature.Whole mount lacZ histochemistry was performed on E9.5-E14.5 day old heterozygous in vitro mouse embryo culture technique 2/5% CO2. The culture medium was replaced after 24 hr. After culture for 1 or 2 days, explants were processed for paraffin embedding and H&E staining as described above.For the explant cultures we developed a new serum free culture system that combined a serum-free Gad1 or Viaat heterozygotes. To increase the likelihood that litters were within comparable ranges of gestational age matings were set up at 9pm and the mice were separated at 9am the next day. Muscimol injections were done within the same time window for each set of pregnant dams. This was done to improve the reproducibility of the results between different litters. In the Gad1-/- rescue experiment, muscimol was injected into the abdominal cavity of pregnant dams every 24 hours from E13.5 to E15.5. For the Viaat-/- rescue experiment, muscimol was injected every 12 hours from E13.5 to E16.0. We used 4 mg/kg of muscimol for each injection. This is a dosage that results in obvious sedation of the mice To generate homozygous offspring for the rescue experiments, we intercrossed RT-PCR analysis of mRNA expression was performed as previously described Viaat (578 bp)5′-GTCGAGGGAGACATTCATTATCAG-3′, 5′-GTACACAGCAGACTGAACTTGGAC-3′Gad1 (302 bp)5′-CCTTCGCCTGCAACCTCCTCGAAC-3′, 5′-GCGCAGTTTGCTCCTCCCCGTTCTT-3′GAPDH (310 bp)5′-GTCTACATGTTCCAGTATGACTCCACTCAC-3′, 5′-CAATCTTGAGTGAGTTGTCATATTTCTCGT-3′Gabrb3 (356 bp)5′-GAAATGAATGAGGTTGCAGGCAGC-3′, 5′-CAGGCAGGGTAATATTTCACTCAG-3′Ucp-1 (593 bp)5′-TAGGTATAAAGGTGTCCTAGGGA-3′, 5′-CGCTTGGGTACTGTCCTGG-3′Viaat and Gad1 for genetic studies and to facilitate the detection of Viaat or Gad1 expressing cells by β-galactosidase histochemistry. The lacZViaat and lacZGad1 mice add to the existing mouse resources that express lacZ or fluorescent proteins in GABAergic and/or glycinergic neurons .To define the spectrum of mutant phenotypes in the  breathe . All Vial region . HistololacZViaat -/- newborn phenotype to that of lacZGad1-/- newborns. Nearly all (98%) of the newborn lacZViaat homozygotes exhibited umbilical hernias  umbilical hernia phenotype that had not been previously reported. Histological sections of lacZViaat homozygotes revealed that this defect was indeed an umbilical hernia, not an ompahalocele as previously reported for the Viaat homozygous mice Gad1 and Viaat functions are not necessary for body wall formation per se but are instead required for the retraction of the umbilical hernia during development. In both genotypes, palate shelf elevation fails to occur at E14.5 and all Gad1 and Viaat mutants were born with cleft secondary palates . Ucp1 is a specific marker of brown fat lacZViaat homozygotes leads to the displacement of these fat deposits to this abnormal location.We also examined the unusual “bumps” on the dorsal cervical region of the -/- mice . These “Gabrb3 receptor subunit and Gad1 mRNAs are expressed in the developing palate and teeth as well as in a variety of other non-neuronal cell types Viaat function is required in the palate or craniofacial structures it should be expressed in these tissues. To determine whether Viaat mRNA is expressed in the developing palate, we examined the expression of Gabrb3, Gad1 and Viaat in mRNA from dissected palatal shelves from E13.5 and E14.5 day old mice. Using RT-PCR we easily detected Gad1 and Gabrb3 transcripts but could not detect Viaat mRNA in this tissue  that expresses Cre under the control of the Nestin regulatory sequences Gad1 used in our experiments has been described previously To perform a genetic test of the requirement for Gad1 we crossed lacZ/+Gad1; NesCre mice to flox/Gad1floxGad1 mice. To confirm that Gad1 had been inactivated specifically in the CNS of the flox/GadlacZGad ; NesCre/+ offspring from this cross we used RT-PCR to measure Gad1 mRNA levels in RNA isolated from dissected palatal shelves and brain from E14.5 embryos. The RT-PCR analysis showed that the amount of Gad1 mRNA in the palatal shelves of the CNS specific Gad1 knockout embryos  was equivalent to controls, while Gad1 mRNA levels in the CNS were greatly reduced as compared to the control embryos . Inactivation of Gabrb3 in mice led to the development of cleft palate in a proportion of the homozygous offspring Gabrb3 was driven by the neuron specific enolase (NSE) regulatory sequences in a genetic background that lacked Gabrb3Gabrb3 function is required in the palate or other non-neural tissues for normal palate development Gabrb3 temporal and spatial expression pattern in the transgenic embryos was provided. This is important since NSE transgenes can be subject to position effects resulting in very large differences in the spatial expression pattern of expression within the CNS between different transgenic lines Gabrb3 was being expressed early enough in the NSE-Gabrb3 transgenic lines tested in this previous study.In contrast, some previously published studies support a developmental function for GABA signaling outside of the CNS. Most of these results came from genetic studies of the gene encoding the β3 subunit of the GABAGabrb3 gene Gabrb3 gene was inactivated specifically in neurons by crossing to a synapsin-Cre transgenic line Gabrb3 specifically in neurons resulted in normal palate development in the mice. Although the synapsin-Cre (SynCre) line is well characterized and has been used to generate neuron specific gene inactivation, the timing of expression may not be ideal for assessing Gabrb3 function in palate formation. The SynCre transgene is specifically expressed in postmitotic neurons and is not widely expressed in the CNS until E13.5 days floxGabrb3 mice the Cre may inactivate the conditional Gabrb3 allele too late in development to interfere with palate elevation and fusion which occur during E13.5-E14.5. This is a particular concern since the authors were relying on the inactivation of two floxGabrb3 alleles in the offspring. Unfortunately, the authors did not monitor Gabrb3 transcript levels in the CNS and palate of the SynCre floxGabrb3/floxGabrb3 mice or the extent of Cre mediated recombination of the Gabrb3flox allele in these experiments A second set of observations supporting a developmental function for GABA in non-neural tissues comes from studies of a neuron-specific knockout of the Fetal akinesia during human or rodent development has been shown to cause defective development of several non-neural tissues and structures in the fetus. For example, it has been shown that normal lung development requires spontaneous fetal breathing movements in humans and mice Gad1 and Viaat knockouts can be understood as additional examples of non-neural phenotypes caused by changes in fetal CNS function that in turn lead to changes in fetal muscle tone or fetal movements. Previous work has shown that fetal movements during palate formation are impaired in the Gad1 mutant mice Gad1 and Viaat homozygous mice are part of a larger spectrum of developmental defects and disorders that are caused by impaired or absent fetal movement. Our work suggests that disruptions of GABA signaling during development could interfere with any one of several developmental processes that depend on fetal movement.Within this context, the craniofacial and body wall phenotypes found in Gad1 or Gabrb3 alleles and human oral clefts Gad1 and Gabrb3 with oral clefts is due to the fundamental requirement for normal GABA neurotransmission for late fetal or neonatal viability. This would eliminate individuals who are homozygous for null or strong loss of function alleles of Gabrb3 or Gad1 from the populations sampled for these studies.In this context is intriguing that there are correlations between the timing of initial fetal movements in mice or humans and the initiation of palatogenesis. In mice, palate elevation and fusion occur at the same time as the first fetal movements are detected Gad1 and Viaat mutant mice are due to a requirement for GABA signaling in the CNS during mouse fetal development. The non-neural defects in these mice are most likely due to defects in fetal movement caused by CNS dysfunction and appear to be part of a spectrum of defects caused by abnormalities in fetal movements. Our results help to clarify the mechanism leading to cleft palate in the Gad1 and Viaat knockout mice and suggest that defects in fetal movement caused by alterations in fetal neuronal GABA signaling may lead to similar defects in humans.In conclusion, our work provides multiple lines of evidence that the non-neural developmental defects in


Nicotinamide and carbogen breathing are both effective radiosensitisers in experimental tumour models and are even more effective in combination. This study was to investigate the feasibility of using the agents in combination in patients and to measure their effect on tumour oxygenation. Twelve patients with advanced malignant disease were treated with 4-6 g of oral nicotinamide (NCT) in tablet formulation. Ten of these 12 patients breathed carbogen  for up to 20 min at presumed peak plasma NCT concentration (Cpeak) and had tumour oxygen partial pressure (pO2) measured using the Eppendorf pO2) histograph. The mean Cpeak values were 82, 115 and 150 micrograms ml-1 for NCT doses of 4, 5 and 6 g respectively and were dose dependent. The time of Cpeak was independent of dose with an overall mean of 2.4 h (range 0.7-4 h). NCT toxicity occurred in 9 out of 12 patients and was mild in all but one; carbogen was well tolerated in all patients. Following NCT only two patients had significant rises (P < 0.05) in tumour median pO2. During carbogen breathing, eight out of ten patients had early highly significant rises in pO2 (P < 0.0001), of which six continued to rise or remained in plateau until completion of gas breathing. Six patients had hypoxic pretreatment values less than 5 mmHg, which were completely abolished in three and reduced in two during carbogen breathing. In conclusion, the combination of NCT and carbogen breathing was generally well tolerated and gave rise to substantial rises in tumour pO2 which were maintained throughout gas breathing. These results should encourage further study of this potentially useful combination of agents as radiosensitisers in the clinic.


In plants, the phytohormone auxin is a crucial regulator sustaining growth and development. At the cellular level, auxin is interpreted differentially in a tissue- and dose-dependent manner. Mechanisms of auxin signalling are partially unknown and the contribution of the AUXIN BINDING PROTEIN 1 (ABP1) as an auxin receptor is still a matter of debate.Here we took advantage of the present knowledge of the root biological system to demonstrate that ABP1 is required for auxin response. The use of conditional ABP1 defective plants reveals that the protein is essential for maintenance of the root meristem and acts at least on the D-type CYCLIN/RETINOBLASTOMA pathway to control entry into the cell cycle. ABP1 affects PLETHORA gradients and confers auxin sensitivity to root cells thus defining the competence of the cells to be maintained within the meristem or to elongate. ABP1 is also implicated in the regulation of gene expression in response to auxin.Our data support that ABP1 is a key regulator for root growth and is required for auxin-mediated responses. Differential effects of ABP1 on various auxin responses support a model in which ABP1 is the major regulator for auxin action on the cell cycle and regulates auxin-mediated gene expression and cell elongation in addition to the already well known TIR1-mediated ubiquitination pathway. TIR1 complex, polyubiquitinylated and degraded via the 26S proteasome abp1 mutant in Arabidopsis ABP1 (At4g02980) exhibit a fairly constant expression in almost all tissues throughout vegetative plant development suggesting that its role is not restricted to embryo development  or via cellular immunization to inactivate ABP1 protein through its in vivo interaction with the recombinant antibody scFv12. The latter recognizes a conformational epitope of ABP1 overlapping the auxin binding site (SS12S and SS12K lines) thus impairing the capacity of the protein to bind and respond to auxin in vivo  and SCARECROW (SCR) are required for QC identity and are also involved in root radial patterning during embryogenesis d plants . Expressd plants . Interes2 mutant . The expPLETHORA family, encoding AP2-domain transcription factors, are also essential for root stem cell maintenance and are related to auxin action. PLTs were recently revealed to control distinct aspects of root development in a dose-dependent manner PLTs was unchanged in roots inactivated for ABP1 but the overall area of expression was consistently reduced compared to controls , thus revealing an insensitivity to auxin of the cells towards the response of division. This data confirms that ABP1 exerts a strict control on cell division and it suggests that ABP1 is required for the capacity of meristematic cells to divide in response to auxin.−6M IAA indicating that cell elongation was not reduced . In the same conditions, Col0 plants exhibited 50% inhibition of growth corresponding to the inhibition of cell elongation. For IAA concentrations higher than 10−6M, a significant inhibition of root growth was observed despite the initial reduced length of ABP1 inactivated roots. This effect resulted from a reduced elongation of the cells as observed for the control at lower concentrations. Roots inactivated for ABP1 were less sensitive than control to primary root elongation inhibition caused by auxin, indicating that ABP1 is also required for this auxin response. Interestingly, it is not a complete insensitivity to auxin as observed for division but a shift in sensitivity suggesting that ABP1 is likely to contribute to this auxin response together with other regulators.Second, we examined the inhibitory effect of exogenous auxin on root elongation. As shown in Aux/IAA mRNA accumulation in response to auxin in short term ethanol induced control and SS12K lines. Auxin efficiently induced the expression of most Aux/IAA genes in root samples with minor changes in comparison to controls as illustrated for IAA3 (out of 14 genes tested) with the notable exception of IAA5, IAA6 and IAA19 . Roots of rRBr and rRBr,SS12K plants were fixed on acetic acid and methanol. Periodic acid-Schiff's reagent was used to reveal starch granules and tissues were stained with Propidium Iodide before observation. The pictures were shaped and assembled using Photoshop (Adobe) without treatment. Quantitative measurements were realised with ImageJ.The β-glucuronisade (GUS) assays were performed as described Immunolocalization was performed on 4 day old seedlings. Immunolocalization in roots was performed as described 13C6]-IAA  internal standard for 2 min at a frequency of 30 Hz, using a Retsch MM 301 vibration mill  and a 3 mm tungsten carbide bead. The samples were then incubated for 15 min at +4°C under continuous shaking. The pH was adjusted to 2.7 with 1 M HCl, and the samples were purified by solid phase extraction on a 500 mg Isolute C8 (EC) column , conditioned with 2 ml methanol and 2 ml 1% acetic acid. After sample application, the column was first washed with 2 ml 10% methanol in 1% acetic acid and then eluted with 2 ml 70% methanol in 1% acetic acid. The dried samples were dissolved in 0.2 ml 2-propanol and 1 ml dichloromethane and 5 µl 2 M trimethylsilyl-diazomethane in hexane (Aldrich) was added to methylate the samples. After methylation, the samples were trimethylsilylated and IAA was quantified by gas chromatography-selected reaction monitoring-mass spectrometry as described in Root tips were collected from 4 dpg seedlings exposed to ethanol for the indicated time and frozen immediately in liquid nitrogen. The frozen samples were homogenized in 0.5 ml 50 mM sodium-phosphate buffer pH 7.0 containing 0.02% diethyldithiocarbamic acid (Sigma) and 500 pg [RNA was extracted from roots of 4dpg seedlings treated with 5 µM NAA for 30 min to 6 hours using an Qiagen RNeasy kit and digested with RNAse free DNAse on the column following the manufacturer's instructions . First-strand cDNAs were synthesized from 5 µg of total RNA using Superscript II reverse transcriptase according to the manufacturer's instructions. Quantitative RT-PCR analyses were performed using SYBR Green QPCR master mix (Roche) with specific primers as reported Figure S1http://www.weigelworld.org/resources/microarray/AtGenExpress/AtGE_dev_samples.pdf/view).Expression pattern of ABP1 in Arabidopsis Expression data were extracted from AtGenExpress developmental series (1.06 MB PDF)Click here for additional data file.


In Arabidopsis, the NRT1 family has eight functionally characterised members and predominantly comprises low-affinity transporters; the NRT2 family contains seven members which appear to be high-affinity transporters; and there are two NRT3 (NAR2) family members which are known to participate in high-affinity transport. A modified reciprocal best hit (RBH) approach was used to identify putative orthologues of the Arabidopsis NRT genes in the four fully sequenced grass genomes . We also included the poplar genome in our analysis to establish whether differences between Arabidopsis and the grasses may be generally applicable to monocots and dicots. Our analysis reveals fundamental differences between Arabidopsis and the grass species in the gene number and family structure of all three families of NRT transporters. All grass species possessed additional NRT1.1 orthologues and appear to lack NRT1.6/NRT1.7 orthologues. There is significant separation in the NRT2 phylogenetic tree between NRT2 genes from dicots and grass species. This indicates that determination of function of NRT2 genes in grass species will not be possible in cereals based simply on sequence homology to functionally characterised Arabidopsis NRT2 genes and that proper functional analysis will be required. Arabidopsis has a unique NRT3.2 gene which may be a fusion of the NRT3.1 and NRT3.2 genes present in all other species examined here. This work provides a framework for future analysis of NO3− transporters and NO3− transport in grass crop species.A large proportion of the nitrate (NO An important first step towards improving the NO3− uptake capacity and the NUE of crop plants would be characterisation of the transporters responsible for NO3− transport. Either the expression of the relevant genes or else the function of the proteins encoded by the genes could then be manipulated through traditional plant breeding or genetic engineering in order to improve NO3− uptake characteristics. With this goal in mind, the aim of this research was to identify the NO3− transporters in grass species.Nitrogen use efficiency (NUE) in plants is determined by the efficiency with which the plant acquires and uses nitrogen. Nitrate  family genes 3−. The NRT1 family comprises predominantly low-affinity NO3− transporters, with the exception of AtNRT1.1 which appears to mediate dual-affinity NO3− transport 3− sensor AtNRT1.2 is constitutive and located predominantly in the root epidermis indicating that the encoded transporter may also be involved in NO3− uptake from the soil AtNRT1.3 in roots is repressed by exposure to NO3− and is induced by NO3− deprivation; its functional role, however, remains less clear AtNRT1.4 is expressed primarily in the leaf petiole and appears to be involved in NO3− storage 3− efflux and to have a role in the loading of NO3− into the xylem for transport to the shoot 3− from maternal tissue to developing embryos 3− from older to younger leaves through facilitating phloem loading 3− from the xylem parenchyma in the roots and shoots, thus working synergistically with AtNRT1.5 to control long-distance NO3− transport.The transport of NONRT2 family are high-affinity NO3− transporters comprising NO3− inducible and constitutively expressed members AtNRT2.1 and AtNRT2.2, which are located next to each other on chromosome 1 and appear to encode proteins with similar function 3− influx and is expressed in the root cortex and epidermis AtNRT2.1 and AtNRT2.2, however, are inducible by provision of NO3− to NO3− starved plants AtNRT2.3 other than its expression may increase and decrease in cycles over the life cycle in the roots and shoots (mostly shoot) AtNRT2.4 is expressed predominantly in the root, and expression appears to decrease following exposure of plants to NO3−AtNRT2.5 is expressed in the root and shoot (mostly root) and is repressed by the provision of NO3−AtNRT2.6 remains relatively unchanged in roots and shoots (mostly root) following exposure of plants to NO3−3− in seeds The NRT3 genes in Arabidopsis play a role in NO3− transport through regulating the activity of NRT2 genes, but are not themselves transporters NRT3 genes appear to be closely related, but NRT3.1 (NAR2.1) appears to play the more significant role in high-affinity NO3− uptake AtNRT3.2 gene is larger than originally published http://www.arabidopsis.org/).The NRT1, NRT2 and NRT3 gene families in the four fully sequenced grass genomes of rice Brachypodium3− transporters in grass species. The evolution from a common ancestor of the four species studied is such that they provide a good indication of the diversity of genomes within the grass species; maize and sorghum are the most closely related, having diverged an estimated 12 million years ago NRT genes is presented.Here, bioinformatic analyses are presented of the A commonly accepted, extensively used and well documented method for the determination of genes that share a common evolutionary ancestor  across two genomes is the reciprocal best hit (RBH) NRT1 genes from the PTR genes (the protein products of which transport peptides) are unknown, the analysis here was limited to determining putative grass orthologues of eight functionally characterised NRT1 genes  genes and the complexity of ancestral events, rendered clear orthologous relationships between dicots and monocots, unattainable.As the genetic sequence(s) or protein motif(s) that separate the T1 genes . ResultsNRT1 genes was less than 10−139, with AtNRT1.1 – AtNRT1.5 and AtNRT1.8 showing greater sequence similarity to their grass counterparts  than AtNRT1.6 and AtNRT1.7 (≈50% sequence identity across ≥80% of the query sequence). Furthermore, RBH analyses returned near identical hits to the grass genomes for AtNRT1.5 and AtNRT1.8 as well as for AtNRT1.6 and AtNRT1.7. Graphical representation of the dicot – monocot RBH analysis can be seen in NRT1 family is illustrated in RBH BLASTp E values in both forward and reverse direction for all eight NRT1 genes and their identified grass homologues . As the genes fall into three subclades, it is likely that gene duplication events gave rise to the three groups after the dicot-monocot split. For AtNRT1.2, AtNRT1.3 and AtNRT1.4 we see a much clearer picture, all cluster with a single clade of grass genes containing at least one member from the four grass species (rice and maize have an extra representative in the 1.3 and 1.4 clade respectively). Notably, poplar has no AtNRT1.4-like gene. AtNRT1.5 and AtNRT1.8 sit together with two and one poplar orthologues respectively and branch off from the main tree with two separate but complete clades of grass genes. Due to a slightly higher dicot – monocot RBH score for 1.5 over 1.8, the grass members of these two clades were named 1.5A and 1.5B. Upon close scrutiny of this branch, we found that the grass 1.5B clade had a nearer phylogenetic neighbour (a PTR gene – At5g19640) even though BLAST result scores were higher for 1.5 and 1.8. Since both clades sit equidistant from AtNRT1.5 (and neither more closely related to AtNRT1.8) an unambiguous assignment of orthology is not possible. Similarly, AtNRT1.6 and AtNRT1.7 sit together on the tree but unlike 1.5 and 1.8, 1.6 and 1.7 share one poplar orthologue and branch of the main tree with a single clade of grass genes missing both a maize and sorghum representative. Interestingly, the sorghum genome was found to contain a degraded pseudogene version that is related to NRT1.6 and NRT1.7, but this transcript is likely to be non-existent as no ESTs exist in any database.The phylogenetic tree of the eight Arabidopsis mologues  depicts Brachypodium - were initially included in the potential orthologue list after RBH analysis but rejected after refinement and validation. These sequences were not included as they were clearly seen to be nearest neighbours to three Arabidopsis PTR genes . Results of the best hits for AtNRT2.5 were marginally lower . Graphical representation of the dicot – monocot RBH analysis can be seen in NRT2 family is illustrated in The seven members of the 1 family . RBH anaNRT2 family painted a particularly interesting picture, exclusive of NRT2.5, the grass NRT2 genes sit entirely separate on the phylogenetic tree from the Arabidopsis NRT2 genes  cluster with the Arabidopsis sequences suggesting that the NRT2 genes developed primarily following the divergence of the monocots and dicots. Thus identification of a clear grass orthologues was only achieved for AtNRT2.5. A close investigation of the genomic localisation of these genes revealed clustering of the genes somewhat reminiscent of genes involved in disease resistance. AtNRT2.1 and AtNRT2.2 are neighbouring genes in opposing orientation, AtNRT2.3 and AtNRT2.4 are tandem repeats and AtNRT2.6  was located on a completely separate chromosome to the others. Correspondingly, of the twelve related sequences in grass , while Brachypodium has two sets of closely located NRT2 genes which are immediately adjacent to each other. Interestingly, the pair of NRT2 genes found in each of the grass species is generally separated by a non-NRT2 gene. These non-NRT2 genes do not share any sequence similarity with one another, nor to any other functionally characterised proteins in the databases (BLASTx search against NCBI). The pairs of NRT2 genes in the grass species may have similar function to AtNRT2.1 and AtNRT2.2, although confirmation of this would require proper functional analysis. Analysis of the gene structure of the NRT2 genes showed that the dicot NRT2 genes all contain both exons and introns, while none of the grass NRT2 genes contain introns  (AtNRT3.2), the proteins translated from the other two splice forms  are 311aa in length, the first 281 aa being identical with that of the longest splice form. Furthermore, there is evidence (NCBI Acc# DQ492237) for another splice product from this locus, which gives rise to a protein of 209 aa overlapping with the C-terminal half of the longest splice form. This protein sequence (referred to here as NRT3.2CT) was described and compared to NRT3.1 by Okamoto et al Genome analysis revealed the 4G24730)  and S5. NRT3.1 but all three of these translate into the same protein (http://arabidopsis.org/servlets/TairObject?type=gene&name=AT5G50200.3).When the protein sequence of At4G24730.1 was used in BLAST searches it became apparent that in the other species in this study the N- and C terminal halves of At4G24730.1 are coded for by two distinct genes. Consequently, it was decided that the protein sequences of NRT3.2SF2/3 and NRT3.2CT would be used separately for orthology searches. Notably, three splice forms have been predicted for Brachypodium  compared with NRT3.2 (≈68%)  and sorghum (Sb07g024380.1), but not in rice. E values, sequence identity and alignment length for these hits were found to be just below those of the top hits . Although considered noteworthy, the maize and sorghum sequences were not used in any subsequent analysis. Similarly, AtNRT3.2 and AtNRT3.2SF2/3 produced the same RBH results, identifying a single NRT3 orthologue in maize, sorghum and rice and a tandem repeat in hypodium . BLAST ENRT3 loci in Arabidopsis lyrata and in a further four dicot species each with a fully sequenced genome. As can be seen from Arabidopsis species is the organisation such that a di-cistronic mRNA can give rise to a fusion product through alternative splicing. In Ricinus communis, Vitis vinifera and Poplar trichocarpa the NRT3.2SP2/3 gene and the NRT3.2CT gene are encoded on the opposite strand of the double helix, and no additional separate NRT3.1-like gene could be found. The exception is Medicago trunculata which appears to lack any NRT3.2CT (or NRT3.1) orthologues in the genome at all. This organisation, with the genes being encoded on opposite strands in direct genomic vicinity, is true also for Carica papaya, Cucumis sativus, and Manihot esculenta (data not shown). These results clearly indicate that the genomic organisation found in Arabidopsis may be the exception rather than the rule for dicots.An attempt was made to explain our finding that At4G24730.1 appeared to be a fusion of what seemed to be two distinct genes in other species. This was done by comparing the genomic organisation of the NRT genes. Several studies have considered the NRT genes in grasses, but have resulted in some confusion, not least in the nomenclature ascribed to the various NRT genes identified. Much of this confusion was due, presumably, to the unavailability of fully sequenced grass genomes leading, for example, to the cloning of orthologues using degenerate primers. For instance, Lin et al NRT gene in rice which was referred to as OsNRT1.1 by Tsay et al NRT1(PTR) family, but it is not a likely orthologue for AtNRT1.1. From the present analysis, and that of Tsay et al AtNRT1.1 than Os03g13274 as originally suggested by Lin et al AtNRT1.1; however this gene does not share as high a similarity with AtNRT1.1 as do the four genes identified in our analysis .The purpose of this study was to determine the cereal orthologues of the characterised Arabidopsis NRT family structure between Arabidopsis and the grasses indicate that Arabidopsis may not be the best model for interpreting NO3− transport in the grasses. The grasses have 3–4 closely related co-orthologues to AtNRT1.1. Recent work indicates that AtNRT1.1 (CHL1) may function as a nitrogen sensor Oryza sativa Build #80 (http://www.ncbi.nlm.nih.gov/UniGene/UGOrg.cgi?TAXID=4530) shows that OsNRT1.1A and OsNRT1.1B are expressed throughout the rice plant, however OsNRT1.1A is predominantly expressed in the root and is expressed more highly than OsNRT1.1B. This indicates that at least two genes potentially fill the same functional role of AtNRT1.1 in grasses. Conversely, the grass genomes lack certain NRT genes that have been characterised in Arabidopsis. Our analysis reveals that the Brachypodium and rice genomes contain only one protein similar to AtNRT1.7 and AtNRT1.6, or no orthologues in the case of maize and sorghum. AtNRT1.6 appears to be involved in transport of NO3− from the maternal tissue to the developing embryo 3− from the older leaves Oryza sativa Build #80 shows OsNRT1.7 is expressed in flower, seed and panicle, perhaps indicating that NRT1.7 in the grasses fills a similar functional role to AtNRT1.6. Whether the proposed long distance NO3− transport functions of the AtNRT1.5 OsNRT1.5A is expressed predominantly in root and panicle, but also in stem, leaf and flower. OsNRT1.5B was mostly expressed in panicle, but also flower and stem.The important differences which exist in NRT2 gene family. The presence of closely located NRT2 genes is reminiscent of the AtNRT2.1/AtNRT2.2 cluster in Arabidopsis and functional analysis of these genes may show similar function between the NRT2 gene clusters. However, with extra duplication in Brachypodium, for example, it will be interesting to identify the functional role played by each of the repeats. Since the NRT2 genes in the grasses lack an intron, it would appear that development of the NRT2 family occurred following the split between the dicots and monocots. It is possible that when they diverged from primitive dicots, the early monocots lost the NRT2 intron(s) whilst the dicots retained the intron(s). However, determination of the development of the NRT2 gene family in plants requires significant further analysis including the genomes of species all along the evolutionary tree, especially other monocots and will need to wait until more genomes are fully sequenced. The NRT2 family is part of the Major Facilitator Superfamily (MFS); of interest, therefore, would be an investigation of the other gene families in this superfamily to establish whether they show the same dichotomy in exon/intron structure. The grass genomes do not contain an AtNRT2.7 orthologue. Since the function of this gene is to load NO3− into the seeds of Arabidopsis 3− into seeds and embryos (similarly to the lack of AtNRT1.6 described above). Further analysis is required to determine whether the grass NRT2 genes have a similar function to that of the Arabidopsis NRT2 genes, or whether their evolutionary divergence also results in a divergence in function. Should there be a divergence in function the isolation of any common protein motifs or sequence differences that separate the dicot and grass NRT2s may provide important structure/function information of value in guiding biotechnological approaches to improving NO3− transport in plants. Although not included in our full analysis (since the genome has not been fully sequenced) the four barley (Hordeum vulgare) NRT2s Brachypodium NRT2 genes (BdNRT2.1/BdNRT2.2 and BdNRT2.3/BdNRT2.4)  ; consequAtNRT3.2SF2/3 gene with the AtNRT3.1CT gene in Arabidopsis compared with the way this protein interacts with the high affinity NRT2 transporters in grasses remains unknown. It will be interesting to determine whether the AtNRT3.2SF2/3 orthologues in the grasses are involved in NO3− transport. In barley (Hordeum vulgare), three NRT3 genes  have been identified AtNRT3.1 or AtNRT3.2CT genes . We have attempted to take this issue into account by naming all potential co-orthologues A, B, C, etc. However, in the case of the NRT2 gene family where dicot and grass genes do not cluster together in the phylogenetic tree, this approach becomes problematic. Therefore, we named family members NRT2.1, NRT2.2, etc, despite the fact that a grass NRT2.1 may not share a functional role with AtNRT2.1.To assist future research we have developed a nomenclature for grass RT genes . WithoutNRT gene families in Arabidopsis and the grasses has revealed some striking differences in gene family structure. Important questions about the evolution of NRT transporters in plants and, significantly, about the suitability of Arabidopsis as a model for NO3− transport in the grasses have also been posed. With the current exponential increase in the availability of molecular genetic resources for cereal crop plants it appears likely that the relevance of Arabidopsis research will decline. This analysis provides a framework for future studies of NO3− transporters and transport in the grasses, and potentially will guide strategies for improvement of NUE in cereal species through genetic manipulation of the NRT genes.In conclusion, the present analysis of the AtNRT family members . The complete database of predicted amino acid sequences from Arabidopsis thaliana and Populus trichocarpa as well as from four monocot species; Zea mays, Oryza sativa, Brachypodium distachyon and Sorghum bicolor were downloaded from public databases (DNA and amino acid sequences of 17  members . were reatabases .Brachypodium and sorghum). This was achieved by using BLASTp, standalone version 2.2.21 Identification of homologues was based primarily on sequence similarity between the 17 Arabidopsis NRTs and the predicted amino acid sequences of the four above mentioned monocots  http://tcoffee.vital-it.ch/). Trees were built using various programs of the Phylogenetic Interference Package (PHYLIP) 3.63 . One thousand bootstrap datasets were generated with SEQBOOT to estimate the confidence limits of nodes. Protein distance matrices were calculated with PROTDIST using the PMB model Geneious v4.8 . Information on genome organisation was obtained from the Phytozome (www.phytozome.org) and TAIR.The RBH obtained sequences for NRT1, were aligned by MAFFT version 6.240 using the L-INS-I method with associated default parameters at  poplar, (B) Brachypodium, (C) maize, (D) sorghum and (E) rice. BLASTp hits of equal e‐value in forward and reverse directions are depicted as forward facing and reverse facing arrows respectively. The colour code of all arrows represents the order of hits, as returned by the BLAST program, first best hit (red), second best hit (blue), third best hit (orange), fourth best hit (green), fifth best hit (black). Truncated arrows indicate a minor drop in the e‐value score between hits. Also coded are the gene accession numbers; an asterisk (*) indicates the presence of alternative splice forms all scoring identical BLASTp results, an upward facing arrow head (∧) indicates a tandem repeat with identical BLAST score, red accessions indicate neighbouring genes in opposing orientation with identical BLAST scores and blue accessions indicate genes in close proximity to each other with identical BLAST scores.(TIF)Click here for additional data file.Figure S2Phylogenetic relationship of potential grass PTR transporters orthologues to AtNRT1.6 and AtNRT1.7. Unrooted Neighbour‐joining tree of NRT1 transporters in Arabidopsis (black), poplar (blue) and 4 grass species: rice (orange), sorghum (green), maize (red) and Brachypodium (purple). Highlighted in the box are the three closest PTR homologues to AtNRT1.6 and AtNRT1.7  as well as orthologous grass PTR transporters . This figure provides rationale for exclusion of grass transporters (brown) as orthologues to AtNRT1.6 and AtNRT1.7. Bootstrap values from 1,000 replicates were used to estimate the confidence limits of the nodes. The scale bar represents a 0.2 estimated amino acid substitution per residue.(TIF)Click here for additional data file.Figure S3Conservation of closely localisedNRT2genes. Gene identifiers and chromosome number are provided for NRT2 genes (red) for (A) Arabidopsis, (B) poplar, (C) rice, (D) Brachypodium, (E) maize and (F) sorghum. Sorghum has a third closely localised NRT2 gene (blue). Also depicted are non‐NRT2 genes (black) between NRT2 genes. Illustrations are not to scale.(TIF)Click here for additional data file.Figure S4Depiction of AtNRT3 genes. Schematic of the AtNRT3.2 locus (AT4G24730) from TAIR 9 GBrowse (http://gbrowse.arabidopsis.org/). Represented are chromosome, BAC, locus, gene models from TAIR 8 and TAIR 9 and cDNA details.(TIF)Click here for additional data file.Figure S5Alignment of AtNRT3 proteins. Proteins included are AtNRT3.1 and AtNRT3.2CT described previously Okamoto et al (TIF)Click here for additional data file.Figure S6Phylogenetic relationship of the NRT2 family including barley family members. Unrooted Neighbour‐joining tree of NRT2 transporters in Arabidopsis (black), poplar (blue) and 5 grass species: rice (orange), sorghum (green), maize (red), Brachypodium (purple) and barley (brown). The four barley members include HvNRT2.1 (HVU34198), HvNRT2.2 (HVU34290), HvNRT2.3 (AF091115) and HvNRT2.4 (AF091116). Bootstrap values from 1,000 replicates were used to estimate the confidence limits of the nodes. The scale bar represents a 0.08 estimated amino acid substitution per residue.(TIF)Click here for additional data file.Figure S7Phylogenetic relationship of the NRT3.1 or NRT3.2CT family including barley family members. Unrooted Neighbour‐joining tree of NRT3.1 or 3.2CT family in Arabidopsis (black), poplar (blue) and 5 grass species: rice (orange), sorghum (green), maize (red), Brachypodium (purple) and barley (brown). The three barley members include HvNRT3.1A (HvNAR2.1 ‐ AY253448), HvNRT3.1B (HvNAR2.2 ‐ AY253449) and HvNRT3.1C (HvNAR2.3 ‐ AY253450). Bootstrap values from 1,000 replicates were used to estimate the confidence limits of the nodes. The scale bar represents a 0.09 estimated amino acid substitution per residue.(TIF)Click here for additional data file.Table S1Identifiers, annotations and family similarities for the ArabidopsisNRT genes described in this study. Provided are the TAIR annotation and identifier, UniprotKB identifier and RefSeq identifier for each NRT gene. Amino acid length of each protein is provided as well as the amino acid identity (%) between the NRTx.1 protein and the other family members.(XLS)Click here for additional data file.Table S2Comparison of theNRT2gene intron and exon numbers for dicots and grasses. Gene identifiers and exon and intron numbers are provided for the dicots Arabidopsis thaliana, Populus trichocarpa, Manihot esculenta, Ricinus communis and Vitis vinifera; and for the grasses Oryza sativa, Zea mays, Sorghum bicolor and Brachypodium distachyon.(XLS)Click here for additional data file.Table S3Web addresses for the genome databases searched during this study including Arabidopsis thaliana, Brachypodium distachyon, Oryza sativa, Populus trichocarpa, Sorghum bicolor and Zea mays.(XLS)Click here for additional data file.Table S4List of candidate homologues excluded from analysis after manual inspection of multiple sequence alignments and their corresponding trees.(XLS)Click here for additional data file.


P and PtdInsP2, are present on intracellular endosomal compartments, and several studies suggest that they have a role in membrane remodeling and trafficking. We refer to them as ‘endosomal PIs’. An increasing number of human genetic diseases including myopathy and neuropathies are associated to mutations in enzymes regulating the turnover of these endosomal PIs. The PtdIns3P and PtdInsP2 3-phosphatase myotubularin gene is mutated in X-linked centronuclear myopathy, whereas its homologs MTMR2 and MTMR13 and the PtdInsP2 5-phosphatase SAC3/FIG4 are implicated in Charcot–Marie–Tooth peripheral neuropathies. Mutations in the gene encoding the PtdIns3P5-kinase PIP5K3/PIKfyve have been found in patients affected with François–Neetens fleck corneal dystrophy. This review presents the roles of the endosomal PIs and their regulators and proposes defects of membrane remodeling as a common pathological mechanism for the corresponding diseases.Phosphoinositides (PIs) are lipid second messengers implicated in signal transduction and membrane trafficking. Seven distinct PIs can be synthesized by phosphorylation of the inositol ring of phosphatidylinositol (PtdIns), and their metabolism is accurately regulated by PI kinases and phosphatases. Two of the PIs, PtdIns3 P and PtdInsP2 are involved in membrane trafficking P2. PtdIns3P was first discovered through the characterization of PI kinases in fibroblasts and by radioactive labeling of astrocytoma cells, while studies in osmotically stressed yeast strains and in resting mouse cells led to the detection of in vivo PtdInsP2 pools P2 metabolism unbalance on the phenotypic manifestations remains poorly understood, but one may hypothesize that the diseases share some common pathomechanisms involving abnormal trafficking. Phenotypes seen in patients may thus highlight new roles of PIs and implicated regulators, and conversely, the known roles of PIs and regulators may help to understand the pathophysiology of the related diseases.In eukaryotic cells, spatiotemporal regulation of cellular organization and cell–cell communication require tightly regulated second messengers and membrane microdomains. Phosphoinositides (PIs) are lipid second messengers implicated in signal transduction and membrane trafficking through recruitment of protein effectors to their site of action . Phosphand PtdIns,5P2 are P2 pools –7. TheirP by the class III PI 3-kinase . PtdIns3P is subsequently phosphorylated by the type III PI 5-kinase  into PtdInsP2, the reverse reaction being catalyzed by the phosphatase SAC3 (the ortholog of S. cerevisiae FIG4). PtdInsP2 can be dephosphorylated by the 3-phosphatase myotubularins (MTMs), leading to the production of PtdIns5P. Myotubularins also dephosphorylate PtdIns3P into PtdIns P2 through the action of class II PI 3-kinase, 4-phosphatase and 5-phosphatase acting on PtdInsP3 or class I PI 3-kinase acting on PtdIns5P, respectively  and can recruit proteins containing FYVE, PX or PH motifs (P (S. cerevisiae) produces PtdIns3P and binds Rab5  against PIK3C3   . A rare ne study ,28. ThisPIP5K3 gene have been found in cases of autosomal dominant François–Neetens corneal fleck dystrophy , which is characterized by abnormal swollen keratocytes with enlarged vesicles of unknown origin in the cornea  retrograde transport and PIP5K3 interacts with p40, a RAB9 effector implicated in retrograde traffic from the late endosome P2 levels increase upon hyperosmotic shock and lead to vacuolar fragmentation P2 effectors  are a heterogeneous group of genetic peripheral neuropathies affecting motor and sensory nerves and are characterized by progressive distal muscle weakness and atrophy. Nerve conduction velocity (NCV) tests are used to differentiate between the demyelinating forms of CMT  and the axonal form of CMT . Autosomal recessive forms of demyelinating CMT are collectively designated CMT4 . Nerve bSAC3 gene have been identified in four unrelated patients with autosomal recessive CMT neuropathy named CMT4J (OMIM 611228) (Compound heterozygous mutations of the human  611228) . These pSAC3 mutations has been allowed by the observation of phenotype similarities between CMT patients and the ‘pale tremor mouse’. This mouse contains an insertion of a transposon into an intron of the SAC3 gene resulting in a loss of the protein and presents neuronal degeneration in the central nervous system, peripheral neuropathy and diluted pigmentation. Cultured fibroblasts from pale tremor mice are filled with enlarged late endosomes/lysosomes and show a reduced level of PtdInsP2 compared with wild type in apparent discrepancy with the PtdInsP2 5-phosphatase function of SAC3 P2 levels P2 was found mutated in autosomal recessive demyelinating neuropathy CMT type 4B  . MTMR2 insP2–60. The proteins ,61.Mtmr2 knockout mouse models develop a mild CMT4B1-like neuropathy and azoospermia. Myelination of peripheral nerves is abnormal in the knockout mice, with myelin outfoldings and recurrent loops originating at paranodal regions , which has similar pathological features to CMT4B1, with additional glaucoma in some patients P2P2 and subsequent membrane trafficking defects.f CMT4B2 ,84. The nsP2. FurthernsP2. In the MTM1) gene have been identified in patients affected with X-linked centronuclear myopathy, also called myotubular myopathy  (MTM1 mutations have been reported in more than 300 unrelated families (Numerous mutations of the myotubularin ( 310400) . XLCNM ifamilies ,87. Theyfamilies . Almost families ,88.Mtm1 knockout mice recapitulate the histopathological signs of XLCNM and show a progressive myopathy starting a few weeks after birth, while muscle histology appears normal at birth  lead to reduced fluid-phase uptake into coelomocytes P2P2. A role nsP2,94. ThusMTM1 and MTMR2. Even if ubiquitous, MTM1 is more expressed in skeletal muscles  with similar enzymatic activity and which are implicated in diseases affecting two different tissues, skeletal muscles for MTM1 in XLCNM and Schwann cells in peripheral nerves for MTMR2 in CMT4B1, as emphasized by corresponding tissue-specific ablation experiments in both knockout mice. A first explanation could consist in differential regulation of expression between  muscles . MTM1 an muscles ,93. Conv muscles ,95. Sugg muscles ,93. SuchP and PtdInsP2 and their regulators all share a common function in membrane dynamics, with a possible implication in membrane fission and retrieval. Altered levels of PtdInsP2 and PtdIns3P, following a loss of the MTMR2/MTMR13 complex in patients affected with CMT4B1/B2, may cause a defective transport of membranes between a storage compartment and myelin sheaths. Membrane fission is essential for membrane retrieval, and fission defects may also be the underlying cause of CMT dominant intermediate type B (CMTDIB) because of missense mutations in dynamin 2 (DNM2), a large GTPase involved in the tubulation of membranes and in the release of newly formed vesicles  that targets membrane proteins for lysosomal degradation through a monoubiquitination mechanism  have recently been identified in patients with an autosomal recessive form of CNM P2 may also have roles independent of membrane traffic and may be found elsewhere than on endosomes. Questions remain about the mechanisms responsible for PtdIns3P- and PtdInsP2-dependent membrane remodeling. Tissue-specific interactors of these endosomal PIs are probably involved in the tubulation and scission of membranes and in the transport of the resulting vesicles to correct locations in cells. Such interactors are interesting candidates for centronuclear myopathies and CMT neuropathies for which several responsible genes still remain to be identified.A significant amount of work has recently highlighted a role for the endosomal PIs in membrane retrieval and their implication in human diseases. This leads to the emerging concept that the associated diseases may be caused by altered membrane remodeling and retrieval at plasma membrane and membrane storage compartments. Additionally, PtdIns3


Patients with high-grade astrocytomas have a poor prognosis. However, considerable variation exists within this group of patients with respect to post-operative survival. In order to determine whether genetic alterations might be of help in subdividing this group, we used allele loss on chromosomes 10 and 17p and epidermal growth factor receptor (EGFR) gene amplification in the tumours as genetic parameters and determined their prognostic value. A series of 47 malignant (grade III and grade IV) tumours were genetically characterised, and four types of tumours were found. Type 1 tumours had loss of heterozygosity on chromosome arm 17p (LOH 17p) as the sole genetic alteration. Patients with this type of tumour were relatively young (mean age 39 years) and had a median survival period of 17 months. Type 2 tumours displayed only allele loss on chromosome 10 (LOH 10), type 3 tumours had LOH 10 + LOH 17p and type 4 tumours contained LOH 10 + EGFR gene amplification. Patients with types 2, 3 and 4 tumours were older  and had a shorter survival  than type 1 patients. Multivariate analysis indicated that the genetic subdivision was a significant prognostic variable. In this study, age proved to be of minor importance with regard to survival. Our study revealed a predominance of frontally located tumours in patients with type 1 tumours, i.e. with LOH 17p only.


We have screened 33 ovarian tumours of various grades and stages for the loss of heterozygosity (LOH) of markers on chromosome 9. LOH was detected in 26 cases (79%). Eleven tumours (33%) showed LOH of all informative markers. The remaining 15 cases had partial deletions. Of these, six (18%) had losses on 9p only, three (9%) had LOH confined to 9q and six (18%) had losses on both chromosome arms, four of which had a retention of hetereozygosity in between. There was no association between tumour grade stage or histopathology and any losses. High-density deletion mapping was carried out in 12 selected cases that had partial deletions of 9p and/or 9q. The deleted region on 9p included the cyclin-dependent kinase inhibitor 2 (CDKN2) locus and one tumour was found to have a homozygous deletion of CDKN2. LOH on 9q extended over a larger region. We found evidence for two regions of deletion on 9q, one at 9q34 and the other encompassing the nevoid basal cell carcinoma (Gorlin) syndrome locus on proximal 9q.


Plasmodium falciparum antigenic variation facilitates long-term chronic infection of the host. This is achieved by sequential expression of a single member of the 60-member var family. Here we show that the 5′ flanking region nucleates epigenetic events strongly linked to the maintenance of mono-allelic var gene expression pattern during parasite proliferation. Tri- and dimethylation of histone H3 lysine 4 peak in the 5′ upstream region of transcribed var and during the poised state , ‘bookmarking’ this member for re-activation at the onset of the next cycle. Histone H3 lysine 9 trimethylation acts as an antagonist to lysine 4 methylation to establish stably silent var gene states along the 5′ flanking and coding region. Furthermore, we show that competition exists between H3K9 methylation and H3K9 acetylation in the 5′ flanking region and that these marks contribute epigenetically to repressing or activating var gene expression. Our work points to a pivotal role of the histone methyl mark writing and reading machinery in the phenotypic inheritance of virulence traits in the malaria parasite.In the human malaria parasite  Plasmodium falciparum during the proliferation phase in red blood cells of its human host depends on the successive expression of variant molecules on the surface of the infected erythrocytes. This variation is mediated by the differential expression of a polymorphic parasite protein, P. falciparum erythrocyte membrane protein 1, which is encoded by ∼60 var genes  and upsD types (var1csa) , non-transcribed or poised state (schizont stage) and silent states of the same var gene in its natural chromosomal context  to be active or stably repressed  along a region of 10 kb comprising exon 1 (8 kb) and 2 kb upstream sequences  and 5′-rapid amplification cDNA end (5′-RACE). For this purpose a first strand cDNA was synthesized from total RNA obtained from chondroitin sulphate A (CSA) and CD36 parasite population using a gene-specific primer (D reverse primer) (var2.4as followed by nested PCR using the gene-specific primer var2.3as and the first PCR as template generated a product around 200 bp (var2CSA gene in parasites in which the gene is either repressed or active.To map the histone modification patterns at the equences . Examina primer) . Then a d 200 bp . The PCRd 200 bp . Our resquantitative Chromatin Immunoprecipitation (qChIP) analyses on var2csa loci in CSA parasites in which the var2csa is being transcribed (‘ON’ state termed here var2csaON) or transiently repressed (‘POISED’ state termed here var2csaPOISED) and in CD36 parasites in which the var2csa is stably repressed (‘OFF’ state termed here var2csaOFF)  are associated with high levels of methyl marks in H3K9  (We investigated the role of H3K9 trimethylation in the control of antigenic variation. To this end we carried out 2csaOFF) . All expng stage . On the od stage . This is in H3K9   called PfGam here. The analysed position for silent PfGam was not associated with significant levels of H3K9 trimethyl marks.An asexual blood stage gene, such as his mark . We alsovar gene transcriptional control. ChIP analyses with antibodies against trimethylated H3K4 show that there is a prominent localized enrichment of trimethyl H3K4 at the 5′ flanking region of the var2csaON  that is highly transcribed in asexual blood stage parasites  and compared it to parasite chromatin preparations where the var2csa is stably repressed (var2csaOFF). These results show that levels of tri- and in particular dimethylation of H3K4 are higher for var2csaPOISED compared with var2csaOFF  need to be recruited to this site in order to co-ordinate expression of var genes.A previous study demonstrated that P. falciparum. We have initiated experiments to elucidate their role in var gene silencing.Specific histone methyl mark binding chromodomain proteins, such as HP1, may translate this information into stably repressed chromatin . An active var promoter state was artificially mimicked by selecting blasticidin-resistant parasites. As the analysis of the 5′ upstream region is compromised by the sequence homology with numerous var genes, the authors analysed a single region in the BSD gene or silent var genes. Based on their results, they concluded that H3K9me3, but not H3K9me2 or monomethylation, correlates with repression of var genes. However, our data clearly show that most changes in the histone methyl and acetyl marks occur at the 5′ flanking region of var genes, indicating that investigating coding regions gives only a limited insight into critical DNA elements involved in epigenetic regulation of antigenic variation. With regard to H3K9me3, the study of Chookajorn et al. supports our finding, because this mark spreads from the 5′ flanking region into the coding region of var genes.A recent study analysed histone marks associated with silent var gene state is maintained during many cell generations. We hypothesize that cellular memory may be acquired by inheriting a var locus enriched with H3K4me at the 5′ flanking region. Active transcription restarts in the same ‘marked’var gene, possibly by raising histone trimethyl marks again to levels needed for transcription. Dynamic changes of H3 lysine 4 methylation marks in the 5′ flanking region have been observed in higher eukaryotes and yeast and correlated with transcriptional activation or permissive state of genes (The question arises how an active trypanosomes the mutually exclusive transcription of a vsg gene is mediated by a particular subnuclear compartment called the ‘Expression Site Body’ (Our findings may be relevant for a number of other protozoan pathogens that use antigenic variation and in the mammalian neuronal cells that express a single member of the large odorant receptor gene family . For exate Body’ . In mousP. falciparum generates reiterated var gene expression patterns through multiple blood stage cycles. Our data show that H3K4 lysine and H3K9 lysine methylation/acetylation marks at the flanking region of var genes are involved in setting up distinct var gene states and this in a strictly mutually exclusive manner. Future research will focus on the molecular machinery that can read these marks at particular DNA regions of the 5′ upstream region and translate this information into co-ordinated gene expression pattern of var genes.In conclusion, our findings have important implications for the processes that promote immune escape and pathogenesis in human malaria parasite. The presented data overcome a conceptual gap in our understanding of antigenic variation, namely, how P. falciparum blood stage parasites from FCR3 strain and panning assays for selection of FCR3 parasites that transcribed var genes associated with CD36 and CSA binding were performed according to Parasite histones were purified by modification of a previously described procedure . Precipivar2csa at ring stage and between poised and silent var2csa at schizont stage.The chromatin immunoprecipitation assay was performed as described previously . We did


A Cox multivariate model for survival duration identified age , P = 0.004), grade  and any loss of chromosome 10  as the only independent prognostic variables. Specifically, LOH for chromosome 10 was able to identify a subgroup of patients with grade 3 tumours who had a significantly shorter survival time. We conclude that LOH for chromosome 10 is an independent, adverse prognostic variable in high-grade glioma. © 1999 Cancer Research CampaignLoss of heterozygosity (LOH) for chromosome 10 is the most frequent genetic abnormality observed in high-grade gliomas. We have used fluorescent microsatellite markers to examine a series of 83 patients, 34 with anaplastic astrocytoma (grade 3) and 49 with glioblastoma multiforme (grade 4), for LOH of chromosome 10. Genotype analysis revealed LOH for all informative chromosome 10 markers in 12 (35%) of patients with grade 3 and 29 (59%) grade 4 tumours respectively, while partial LOH was found in a further eight (24%) grade 3 and ten (20%) grade 4 tumours. Partial LOH, was confined to the long arm (10q) in six and the short arm (10p) in three cases, while alleles from both arms were lost in four cases. Five tumours (one grade 3 and four grade 4) showed heterogeneity with respect to loss at different loci. There was a correlation between any chromosome 10 loss and poorer performance status at presentation (χ


Growth factor receptor bound (Grb) proteins 7, 10 and 14 are a family of structurally related multi-domain adaptor proteins involved in a variety of biological processes. Grb7, 10 and 14 are known to become serine and/or threonine phosphorylated in response to growth factor (GF) stimulation. Grb7 and 10 have also been shown to become tyrosine phosphorylated under certain conditions. Under experimental conditions Grb7 is tyrosine phosphorylated by the Tie2/Tie-2/Tek angiogenic receptor tyrosine kinase (RTK). Furthermore, Grb14 has also been shown to interact with Tie2, however tyrosine phosphorylation of this Grb family member has yet to be reported.Here we report for the first time tyrosine phosphorylation of Grb14. This phosphorylation requires a kinase competent Tie2 as well as intact tyrosines 1100 and 1106 (Y1100 and Y1106) on the receptor. Furthermore, a complete SH2 domain on Grb14 is required for Grb14 tyrosine phosphorylation by Tie2. Grb14 was also able to become tyrosine phosphorylated in primary endothelial cells when treated with a soluble and potent variant of the Tie2 ligand, cartilage oligomeric matrix protein (COMP) Ang1.Our results show that Grb14, like its family members Grb7 and Grb10, is able to be tyrosine phosphorylated. Furthermore, our data indicate a role for Grb14 in endothelial signaling downstream of the Tie2 receptor. Growth factor Receptor Bound (Grb) adaptor proteins are a group of structurally similar proteins beginning to emerge as key players in a number of cellular functions including cell metabolism, cell survival and cell migration.Signal transduction pathways encompass a series of highly coordinated events involving numerous proteins, varying in both structure and function. Adaptor proteins play a pivotal role in such molecular networks by allowing formation of protein complexes via interactions involving their non-catalytic binding domains. The Between PH and SH2) domain. To date the BPS domain has been shown to play a role in certain Grb/receptor interactions including those involving the activated Insulin receptor (IR) and Insulin-like Growth Factor Receptor ATP (Amersham Biosciences), 20 μM ATP (Amersham Biosciences) for 30 min at 30°C [ΔPH phosphorylation over the amount of immunoprecipitated Tie2 in each of the samples. All experiments were performed twice or more times with similar results.Kinase assays were performed as follows: Cell lysates were immunoprecipitated with anti-Tie2 antibodies for 2 h at 4°C. Immunoprecipitates were washed 3x in PLC lysis buffer (without sodium fluoride or sodium pyrophosphate)  and 2x i at 30°C . Kinase The authors declare that they have no competing interests.CS performed all the experiments and wrote the manuscript. DD revised the manuscript and provided final approval for publication. All authors have read and approved the final manuscript.


Mutations of the tumour suppressor p53 gene are found in a number of spontaneous canine cancers and may contribute to increased cytogenetic alterations and tumour formation. Using reverse transcription and DNA amplification, we isolated p53 cDNA from normal and tumour tissue of ten canine mammary cancer patients. DNA sequencing identified p53 mutations in three of the ten patients. These included tumour-associated p53 gene mutations within exons 2 and 5 and a germ line deletion of exons 3 to 7. These results support a role for p53 inactivation in canine mammary tumour formation and breed predisposition to cancer. Such information could prove invaluable in the successful outbreeding of inherited predisposition to cancer in the dog. A putative polymorphism was also identified at codon 69 in exon 4 and we discuss the possibility that similar polymorphisms may be associated with human breast cancer. © 1999 Cancer Research Campaign