Differential Display Method Research Articles

Enhanced Expression of Hepatic Genes in Copper-Deficient Rats Detected by the Messenger RNA Differential Display Method

The influence of copper (Cu) status on hepatic gene expression was examined by using the “messenger RNA differential display” technology. This method involves the distribution of mRNA in a two-dimensional array for the rapid identification and cloning of differentially expressed genes. Livers from male Sprague-Dawley rats that had been fed a Cu-deficient (CD) diet (9.4 µmol/kg) or a Cu-adequate (CA) diet (103.9 µmol/kg) for 6 wk were used to supply cytosolic RNA. Cytosolic RNA were reverse-transcribed in the presence of anchor primers and then amplified by polymerase chain reaction with anchor and arbitrary primer sets. The amplified cDNA were then resolved by denaturing polyacrylamide gel electrophoresis. Differences in mRNA expression between the CD and CA rats were identified. DNA fragments were cloned, sequenced and used as probes for Northern blot analysis to confirm that the identified genes were differentially expressed. The analysis of cDNA sequences by computer searches against DNA and protein databases revealed that one cDNA fragment, whose mRNA abundance was enhanced 1.2-fold by copper deficiency, is novel. Four other cDNA fragments were found to have substantial homology with rat ferritin mRNA; rat fetuin mRNA; rat mitochondrial 12S and 16S rRNA, phenylalanine-, valine- and leucine-tRNA genes; rat mitochondrial genes for 16S rRNA, tRNA-leucine and tRNA-valine; and their mRNA abundance was 0.6- to 0.8-fold higher in Cu-deficient rats. Five additional cDNAs detected by this method appeared to represent novel genes because they exhibited no substantial homology to recorded gene and protein sequences deposited in DNA and protein data-bases. These results demonstrate the usefulness of this technology in the detection of genes which were differentially expressed as a result of the deprivation of a single nutrient, dietary copper, in this research project.

Using cDNA SCoT method to identify differentially expressed genes in date palm (Phoenix dactylifera L.)

AbstractDate palm is a relatively salt- and drought-tolerant plant and more recent efforts have been focused on recognizing genes and pathways that confer stress tolerance in this species. The cDNA start codon targeted marker (cDNA–SCoT) technique is a novel, simple, fast and effective method for differential gene expression investigation. In the present study, this technique was exploited to identify differentially expressed genes during several stress treatments in date palm. A total of 12 SCoT primers combined with oligo-dT primers amplified differentially expressed fragments among the stress treatments and control samples. Differentially expressed fragments were highly homologous to known genes or encoded unclassified proteins with unknown functions. The expression patterns of the genes that had direct or indirect relationships with salinity and drought stress response were identified and their possible roles were discussed. This study suggests that the cDNA-SCoT differential display method is a useful tool to serve as an initial step for characterizing transcriptional changes induced by abiotic stresses and provide gene information for further study and application in genetic improvement and breeding in date palm.

Is auxin involved in the induction of genetic instability in barley homeotic double mutants?

The triggers of genetic instability in barley homeotic double mutants are tweaky spike -type mutations associated with an auxin imbalance in separate spike phytomeres. Barley homeotic tweaky spike;Hooded (tw;Hd) double mutants are characterized by an inherited instability of spike and flower development, which is absent in the single parental constituents. The aim of the present study was to show that the trigger of genetic instability in the double mutants is the tw mutations, which are associated with an auxin imbalance in the developing spikes. Their pleiotropic effects on genes related to spike/flower development may cause the genetic instability of double mutants. The study of four double-mutant groups composed of different mutant alleles showed that the instability arose only if the mutant allele tw was a constituent of the double mutants. Application of auxin inhibitors and 2,4-dichlorophenoxyacetic acid (2,4-D) demonstrated the relationship of the instability of the double mutants and the phenotype of the tw mutants to auxin imbalance. 2,4-D induced phenocopies of the tw mutation in wild-type plants and rescued the phenotypes of three allelic tw mutants. The differential display (dd-PCR) method allowed the identification of several putative candidate genes in tw that may be responsible for the initiation of instability in the double mutants by pleiotropic variations of their expression in the tw mutant associated with auxin imbalance in the developing spikes. The results of the present study linked the genetic instability of homeotic double mutants with an auxin imbalance caused by one of the constituents (tw). The genetic instability of the double mutants in relation to auxin imbalance was studied for the first time. A matrocliny on instability expression was also observed.

Molecular characterization of aspartylglucosaminidase, a lysosomal hydrolase upregulated during strobilation in the moon jellyfish, Aurelia aurita.

The life cycle of the moon jellyfish, Aurelia aurita, alternates between a benthic asexual polyp stage and a planktonic sexual medusa (jellyfish) stage. Transition from polyp to medusa is called strobilation. To investigate the molecular mechanisms of strobilation, we screened for genes that are upregulated during strobilation using the differential display method and we identified aspartylglucosaminidase (AGA), which encodes a lysosomal hydrolase. Similar to AGAs from other species, Aurelia AGA possessed an N-terminal signal peptide and potential N-glycosylation sites. The genomic region of Aurelia AGA was approximately 9.8kb in length and contained 12 exons and 11 introns. Quantitative RT-PCR analysis revealed that AGA expression increased during strobilation, and was then decreased in medusae. To inhibit AGA function, we administered the lysosomal acidification inhibitors, chloroquine or bafilomycin A1, to animals during strobilation. Both inhibitors disturbed medusa morphogenesis at the oral end, suggesting involvement of lysosomal hydrolases in strobilation.

FUCA1 is induced by wild-type p53 and expressed at different levels in thyroid cancers depending on p53 status.

Fucose residues of cell surface glycans, which play important roles in growth, invasion and metastasis, are added by fucosyltransferases (FUTs) and removed by α-L-fucosidases (FUCAs). By the differential display method, we isolated a 3'non-coding region of α-L-fucosidase-1 (FUCA1) (a gene coding for the lysosomal fucosidase-1 enzyme) as a wild-type p53-inducible gene: 18S and 20S FUCA1 mRNA species were induced in Saos-2 cells transfected with a temperature-sensitive p53 mutant at the permissive temperature. By microarray analyses of thyroid cancer biopsy samples, FUCA1 RNA expression levels were found to be lower in anaplastic thyroid cancer samples (ATCs), while they were higher in papillary thyroid cancer samples (PTCs) and in normal thyroid tissues. Since most ATCs were reported to carry the mutated form of p53, while PTCs carry mostly the wild-type form of p53, it is likely that FUCA1 expression levels are regulated, at least in part, by the p53 status in thyroid cancers. In order to better understand the role played by FUCA genes in thyroid tumorigenesis, we examined the clonogenic potential invitro of thyroid cell lines transfected with either FUCA1 or FUCA2 (the latter gene coding for a secreted, non-lysosomal enzyme). We found that α-L-fucosidases did not suppress grossly cell growth. Contrary to what we observed with the expression of FUCA1, the FUT8 expression levels were found high in ATCs but lower in PTCs and normal thyroid tissues. Taken together, these results suggest the possibility that the higher fucose levels on cell surface glycans of aggressive ATCs, compared to those of less aggressive PTCs, may be at least in part responsible for the more aggressive and metastatic phenotype of ATCs compared to PTCs, as the expression levels of FUCA1 and FUT8 were inversely related in these two types of cancers.

An evolutionary and developmental approach toward understanding of growth variations of two closely related dwarfism of Galium verum

Galium L. is one of the largest and most widespread genus of Rubiaceae, consisting of more than 650 species worldwide. Galium verum var. asiaticum (G. verum a.) is a perennial herbaceous and widely distributed in in Korea peninsula. On the other hand, Galium verum var. asiaticum for. pusillum (G. verum a.p.) is endemic to Korea, inhabiting only on high land of Mt. Halla of Jeju. G. verum a.p. appears to be a dwarfism of G. verum a. We wondered what physiological, environmental, or genetic factors rendered those two taxa morphologically differentiated. We found that G. verum a.p. shows a low activity of the cell proliferation and was not associated with responsiveness contents of auxin and gibberellins. In order to search for genetic factors involved, we carried out an mRNA differential display method using the ACPs, and isolated several different expression genes between the two taxa. We chose one of those genes, which encoded RADIALIS-like proteins: GvaRADL1 from G. verum a. and GvapRADL1 from G. verum a.p. We discuss the relevancy of the genetic variations in regard to the differential expression patterns of those genes and the differential growth patterns of the two variants.

Characterization of differentially expressed genes in liver in response to the rearing temperature of rainbow trout Oncorhynchus mykiss and their heritable differences.

To characterize thermal-responsive genes in fish, firstly, juvenile rainbow trout were reared in four different temperature conditions (average temperatures were 10, 14, 18, and 22°C, respectively) and differentially expressed genes were identified. Gene expression in the liver was analyzed by the differential display method, followed by validation using real-time PCR. Subsequently, to examine whether the identified genes show heritable differences, the gene expression levels were compared among juveniles of three genetically distinct lines of rainbow trout (a strain and two closed colonies) by rearing at two different temperature conditions (average 14 and 22°C). By rearing at 22°C, growth retardation was observed compared with fish reared at 14 and 18°C, and six genes were identified as differentially expressed genes in response to the rearing temperature in the gene expression analyses. With the increase in rearing temperature, gene expressions of a complement C1q and two ribosomal proteins were significantly up-regulated. On the other hand, three metabolic genes (betaine homocysteine methyltransferase, triosephosphate isomerase, and glucose-6-phosphatase) were down-regulated, indicating a metabolic depression due to high temperature. In the subsequent analyses, in response to the rearing temperature (14 and 22°C), there was a trend that the complement C1q and glucose-6-phosphatase genes showed different expression patterns among the three rainbow trout lines, suggesting heritable differences in these genes. Our study provides information on thermal-responsive genes in fish, and we anticipate it will facilitate further investigation in the thermal biology of fish.

Oligo-dT anchored cDNA-SCoT: a novel differential display method for analyzing differential gene expression in response to several stress treatments in mango (Mangifera indica L.).

Differential display is a powerful technique for analyzing differences in gene expression. Oligo-dT cDNAstart codon targeted marker (cDNA-SCoT) technique is a novel, simple, cheap, rapid, and efficient method for differential gene expression research. In the present study, the oligo-dT anchored cDNA-SCoT technique was exploited to identify differentially expressed genes during several stress treatments in mango. A total of 37 primers combined with oligo-dT anchor primers 3side amplified approximately 150 fragments of 150 bp to 1500 bp in length. Up to 100 fragments were differentially expressed among the stress treatments and control samples, among which 92 were obtained and sequenced. Out of the 92 transcript derived fragments (TDFs), 70% were highly homologous to known genes, and 30% encoded unclassified proteins with unknown functions. The expression pattern of nine genes with known functions involved in several abiotic stresses in other species was confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) under cold (4 °C), salinity (NaCl), polyethylene glycol (PEG, MW 6000), and heavy metal treatments in leaves and stems at different time points (0, 24, 48, and 72 h). The expression patterns of the genes (TDF4, TDF7, TDF23, TDF45, TDF49, TDF50, TDF57, TDF91 and TDF92) that had direct or indirect relationships with cold, salinity, drought and heavy metal stress response were analyzed through qRT-PCR. The possible roles of these genes are discussed. This study suggests that the oligo-dT anchored cDNA-SCoT differential display method is a useful tool to serve as an initial step for characterizing transcriptional changes induced by abiotic stresses and provide gene information for further study and application in genetic improvement and breeding in mango.

Differential susceptibility to hydrogen sulfide-induced apoptosis between PHLDA1-overexpressing oral cancer cell lines and oral keratinocytes: Role of PHLDA1 as an apoptosis suppressor

Hydrogen sulfide (H2S) is a novel gasotransmitter that plays multiple biological roles in various body systems. In addition to its endogenous production, H2S is produced by bacteria colonizing digestive organs, including the oral cavity. H2S was previously shown to enhance pro-apoptotic effects in cancer cell lines, although the mechanisms involved remain unclear. To properly assess the anti-cancer effects of H2S, however, investigations of apoptotic effects in normal cells are also necessary. The aims of this study were (1) to compare the susceptibility to H2S-induced apoptosis between the oral cancer cell line Ca9-22 and oral keratinocytes that were derived from healthy gingiva, and (2) to identify candidate genes involved in the induction of apoptosis by H2S. The susceptibility to H2S-induced apoptosis in Ca9-22 cells was significantly higher than that in keratinocytes. H2S exposure in Ca9-22 cells, but not keratinocytes, enhanced the expression of pleckstrin homology-like domain, family A, member 1 (PHLDA1), which was identified through a differential display method. In addition, PHLDA1 expression increased during actinomycin D-induced apoptosis in Ca9-22 cells. Knockdown of PHLDA1 expression by small interfering RNA in Ca9-22 cells led to expression of active caspase 3, thus indicating apoptosis induction. The tongue cancer cell line SCC-25, which expresses PHLDA1 at a high level, showed similar effects. Our data indicate that H2S is an anti-cancer compound that may contribute to the low incidence of oral cancer. Furthermore, we demonstrated the role of PHLDA1 as an apoptosis suppressor.

Identification of Sec14‐like 3 as a novel lipid‐packing sensor in the lung

Pulmonary surfactant, a complex composed primarily of lipids and associated proteins, is synthesized in alveolar type II (ATII) cells and secreted into alveoli to prevent collapse during respiration. Although numerous studies have clarified the fundamental roles of pulmonary surfactant, the molecular mechanisms of transport and secretion of pulmonary surfactant remain totally unknown. Thus, we screened candidate genes by comparing genes with the expressed sequence tag (EST) libraries of embryonic and adult lungs by using the digital differential display method in the National Center for Biotechnology Information (NCBI) database. We identified Sec14-like 3 (Sec14L3) as a new class of lipid-associated proteins highly expressed in ATII cells. We found that Sec14L3 expression is >100-fold increased during the perinatal period in the lung. Furthermore, Sec14L3 bound to small-sized liposomes (30 nm in diameter), but not to large-sized liposomes (100 nm diameter), through its Sec14 domain. Because of the increased curvature, lipid-packing defects are more likely to occur in small-sized liposomes than in large-sized liposomes. Based on these results, we conclude that Sec14L3 is a new class of lipid-packing sensor. Sec14L3 may play important roles in the lung, such as intracellular lipid transport, surfactant maturation, and endo/exocytosis.

Identification of novel hypoxia response genes in human glioma cell line a172.

Hypoxia is a serious challenge for treatment of solid tumors. This condition has been manifested to exert significant therapeutic effects on glioblastoma multiform or (WHO) astrocytoma grade IV. Hypoxia contributes numerous changes in cellular mechanisms such as angiogenesis, metastasis and apoptosis evasion. Furthermore, in molecular level, hypoxia can cause induction of DNA breaks in tumor cells. Identification of mechanisms responsible for these effects can lead to designing more efficient therapeutic strategies against tumor progression which results in improvement of patient prognosis. Materials and Methods : In order to identify more hypoxia regulated genes which may have a role in glioblastoma progression, cDNA-AFLP was optimized as a Differential display method which is able to identify and isolate transcripts with no prior sequence knowledge. Using this method, the current study identified 120 Transcription Derived Fragments (TDFs) which were completely differentially regulated in response to hypoxia. By sequence homology searching, the current study could detect 22 completely differentially regulated known genes and two unknown sequence matching with two chromosome contig and four sequence matches with some Expressed Sequence Tags (ESTs). Further characterizing of these genes may help to achieve better understanding of hypoxia mediated phenotype change in tumor cells.

Identification and characterization of up-regulated genes in the halophyte Limoniastrum monopetalum (L.) Boiss grown under crude oil pollution

Differential display method was applied to transcripts extracted from leaves of Limoniastrum monopetalum to identify genes that are differentially expressed in response to crude oil pollution. The results showed that 201 bands with different molecular sizes were differentially expressed in polluted plants. Ten cDNA bands were considered to be consistently over-expressed under crude oil stress and selected for sequencing. Comparative analysis of these cDNA sequences allowed us to classify them into six categories: (1) enzymes increase its activity under petroleum stress and were a good marker of petroleum stress (e.g. xanthine dehydrogenase, metallothionein type 2, and arginine decarboxylase), (2) nitrogen metabolism (e.g. glutamine synthetase and amidophosphoribosyltransferase), (3) drought genes (e.g. CPRD2), (4) salinity stress (e.g. retrotransposon protein), (5) plant growth (e.g. aminocyclopropane-1-carboxylic acid and ribulose-1,5-bisphosphate carboxylase oxygenase), and (6) transport related genes like proton-dependent oligopeptide transport (POT) family protein. Coincidently with the differential display results, the amount of the total protein differed significantly between unpolluted and polluted plants ( T = 3.687, P < 0.006). The electrophoretic patterns (SDS–PAGE) for water soluble proteins revealed that 11 peptides with different molecular masses disappeared and eight different peptides were synthesized in polluted plants. The results of up-regulated genes identified in this study may explain the way that L. monopetalum populations established on the crude oil polluted soil and vigorous vegetative growth of adult plants.

Analysis of gene expression profile changes in response to hypoxia using cDNA-AFLP as a differential display method in two human glioma cell lines (U87, A172)

Analysis of gene expression profile changes in response to hypoxia using cDNA-AFLP as a differential display method in two human glioma cell lines (U87, A172)

Identification of differentially-expressed genes potentially related to stress tolerance in a transgenic line of European pear over-expressing an apple spermidine synthase gene (MdSPDS1)

SummaryPolyamines confer abiotic stress tolerance through mechanisms that are not yet fully understood. Previous studies on a transgenic line of European pear (Pyrus communis L., line #32), over-expressing an apple gene for spermidine synthase (MdSPDS1), provided greater insight into the mechanisms of stress tolerance, leading to agronomic benefits, as well as being an elite line to be used to further elucidate the molecular mechanisms underlying stress tolerance that are exerted by spermidine (Spd). In this study, a new and accurate reverse transcription-polymerase chain reaction (RT-PCR) method, involving an annealing control primer (ACP)-based PCR (GeneFishing) system, was employed to identify genes that may be implicated in abiotic stress tolerance. A total of 42 transcripts that were differentially expressed between line #32 and wild-type (WT) pear were identified. After cloning, 24 of these were found to exhibit significant sequence similarities with known genes from other species, and the remaining 18 were annotated as “unknown”. To verify the reliability of the ACP-based differential display method, ten clones (five up-regulated and five down-regulated in line #32 compared to WT) were subjected to real-time RT-PCR analysis. These clones were further analysed to determine the relative abundance of the target sequences under conditions of salinity or cadmium stress. The results indicated that GeneFishing is a useful method to identify differentially-expressed genes (DEGs) in an MdSPDS1-overexpressing transgenic line of pear. The possible involvement of these DEGs in salinity and cadmium stress tolerance is also discussed based on an analysis of their levels of expression.

Identification of differentially expressed genes using an annealing control primer system in stage III serous ovarian carcinoma

BackgroundMost patients with ovarian cancer are diagnosed with advanced stage disease (i.e., stage III-IV), which is associated with a poor prognosis. Differentially expressed genes (DEGs) in stage III serous ovarian carcinoma compared to normal tissue were screened by a new differential display method, the annealing control primer (ACP) system. The potential targets for markers that could be used for diagnosis and prognosis, for stage III serous ovarian cancer, were found by cluster and survival analysis.MethodsThe ACP-based reverse transcriptase polymerase chain reaction (RT PCR) technique was used to identify DEGs in patients with stage III serous ovarian carcinoma. The DEGs identified by the ACP system were confirmed by quantitative real-time PCR. Cluster analysis was performed on the basis of the expression profile produced by quantitative real-time PCR and survival analysis was carried out by the Kaplan-Meier method and Cox proportional hazards multivariate model; the results of gene expression were compared between chemo-resistant and chemo-sensitive groups.ResultsA total of 114 DEGs were identified by the ACP-based RT PCR technique among patients with stage III serous ovarian carcinoma. The DEGs associated with an apoptosis inhibitory process tended to be up-regulated clones while the DEGs associated with immune response tended to be down-regulated clones. Cluster analysis of the gene expression profile obtained by quantitative real-time PCR revealed two contrasting groups of DEGs. That is, a group of genes including: SSBP1, IFI6 DDT, IFI27, C11orf92, NFKBIA, TNXB, NEAT1 and TFG were up-regulated while another group of genes consisting of: LAMB2, XRCC6, MEF2C, RBM5, FOXP1, NUDCP2, LGALS3, TMEM185A, and C1S were down-regulated in most patients. Survival analysis revealed that the up-regulated genes such as DDAH2, RNase K and TCEAL2 might be associated with a poor prognosis. Furthermore, the prognosis of patients with chemo-resistance was predicted to be very poor when genes such as RNase K, FOXP1, LAMB2 and MRVI1 were up-regulated.ConclusionThe DEGs in patients with stage III serous ovarian cancer were successfully and reliably identified by the ACP-based RT PCR technique. The DEGs identified in this study might help predict the prognosis of patients with stage III serous ovarian cancer as well as suggest targets for the development of new treatment regimens.

Gene expression changes during caste-specific neuronal development in the damp-wood termite Hodotermopsis sjostedti

BackgroundOne of the key characters of social insects is the division of labor, in which different tasks are allocated to various castes. In termites, one of the representative groups of social insects, morphological differences as well as behavioral differences can be recognized among castes. However, very little is known about the neuronal and molecular bases of caste differentiation and caste-specific behavior. In almost all termite species, soldiers play defensive roles in their colonies, and their morphology and behavior are largely different from workers (or pseudergates). Therefore, we predicted that some genes linked to defensive behavior and/or those required for neuronal changes are differentially expressed between workers and soldiers, or during the soldier differentiation, respectively.ResultsUsing the brain and suboesophageal ganglion (SOG) of the damp-wood termite Hodotermopsis sjostedti, we first screened genes specifically expressed in soldiers or during soldier differentiation by the differential display method, followed by quantitative real-time polymerase chain reaction. No distinctive differences in expression patterns were detected between pseudergates and soldiers. In the course of soldier differentiation, however, five genes were found to be up-regulated in brain and/or SOG: 14-3-3epsilon, fibrillin2, beta-tubulin, ciboulot, and a hypothetical protein containing a SAP motif. Some of these genes are thought to be associated with cytoskeletal structure or motor-associated proteins in neuronal tissues.ConclusionThe identified five genes could be involved in soldier-specific neuronal modifications, resulting in defensive behaviors in termite soldiers. The temporal expression patterns of these genes were consistent with the neuronal changes during soldier differentiation, suggesting that molecular machineries, in which the identified factors would participate, play important roles in behavioral differentiation of termite soldiers.

Gene Expression of Spag6 in Chick Central Nervous System

Using a differential display method, we identified sperm-associated antigen 6 (Spag6) as a gene with a dynamic expression profile within the chick embryonic spinal cord. The expression of Spag6 gradually decreased along with spinal cord development. Spag6 expression was detected adjacent to the ventricular zone of the spinal cord at embryonic day (E) 4. At E6, Spag6 was apparent in the ventral ventricular zone adjacent to floor plate and the surrounding region close to the ventricular zone, with additional weak expression at the adjacent region to the ventral horn. At E10, the Spag6 mRNA can be detected slightly in the ventral ventricular zone and surrounding region of dorsal ventricular zone. In the E6 hindbrain, Spag6 was detected in the roof, the ventricular zone adjacent to floor plate and the surrounding regions of the ventricular zones. In the E6 caudal diencephalon, Spag6 expression was detected adjacent to the ventricular zone. As Spag6 was expressed in areas containing ependymal progenitor cells and in the borders of undifferentiated regions, Spag6 may be involved in the development of ependymal cells and in the differentiation process of neuronal cells in chick neural organs.

A novel WD40 protein, BnSWD1, is involved in salt stress in Brassica napus

Genes that are expressed early in specific response to high salinity conditions were isolated from rapeseed plant (Brassica napus L.) using an mRNA differential display method. Five PCR fragments (DD1–5) were isolated that were induced by, but showed different response kinetics to, 200 mM NaCl. Nucleotide sequence analysis and homology search revealed that the deduced amino sequences of three of the five cDNA fragments showed considerable similarity to those of β-mannosidase (DD1), tomato Pti-6 proteins (DD5), and the tobacco harpin-induced protein hin1 (DD4), respectively. In contrast, the remaining clones, DD3 and DD2, did not correspond to any substantial existing annotation. Using the DD3 fragment as a probe, we isolated a full-length cDNA clone from the cDNA library, which we termed BnSWD1 (Brassica napus salt responsive WD40 1). The predicted amino-acid sequence of BnSWD1 contains eight WD40 repeats and is conserved in all eukaryotes. Notably, the BnSWD1 gene is expressed at high levels under salt-stress conditions. Furthermore, we found that BnSWD1 was upregulated after treatment with abscisic acid, salicylic acid, and methyl jasmonate. Our study suggests that BnSWD1, which is a novel WD40 repeat-containing protein, has a function in salt-stress responses in plants, possibly via abscisic acid-dependent and/or -independent signaling pathways.

Increased Stathmin1 Expression in the Dentate Gyrus of Mice Causes Abnormal Axonal Arborizations

Pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in multiple brain functions. To clarify the cause of abnormal behavior in PACAP deficient-mice, we attempted the identification of genes whose expression was altered in the dentate gyrus of PACAP-deficient mice using the differential display method. Expression of stathmin1 was up-regulated in the dentate gyrus at both the mRNA and protein levels. PACAP stimulation inhibited stathmin1 expression in PC12 cells, while increased stathmin1expression in neurons of the subgranular zone and in primary cultured hippocampal neurons induced abnormal arborization of axons. We also investigated the pathways involved in PACAP deficiency. Ascl1 binds to E10 box of the stathmin1 promoter and increases stathmin1 expression. Inhibitory bHLH proteins (Hes1 and Id3) were rapidly up-regulated by PACAP stimulation, and Hes1 could suppress Ascl1 expression and Id3 could inhibit Ascl1 signaling. We also detected an increase of stathmin1 expression in the brains of schizophrenic patients. These results suggest that up-regulation of stathmin1 in the dentate gyrus, secondary to PACAP deficiency, may create abnormal neuronal circuits that cause abnormal behavior.

Increased stathmin1 expression in the dentate gyrus of mice causes abnormal axonal arborizations

Pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in multiple brain functions. To clarify the cause of abnormal behavior in PACAP deficient-mice, we attempted the identification of genes whose expression was altered in the dentate gyrus of PACAP-deficient mice using the differential display method. Expression of stathmin1 was up-regulated in the dentate gyrus at both the mRNA and protein levels. PACAP stimulation inhibited stathmin1 expression in PC12 cells, while increased stathmin1expression in neurons of the subgranular zone and in primary cultured hippocampal neurons induced abnormal arborization of axons. We also investigated the pathways involved in PACAP deficiency. Ascl1 binds to E10 box of the stathmin1 promoter and increases stathmin1 expression. Inhibitory bHLH proteins (Hes1 and Id3) were rapidly up-regulated by PACAP stimulation, and Hes1 could suppress Ascl1 expression and Id3 could inhibit Ascl1 signaling. We also detected an increase of stathmin1 expression in the brains of schizophrenic patients. These results suggest that up-regulation of stathmin1 in the dentate gyrus, secondary to PACAP deficiency, may create abnormal neuronal circuits that cause abnormal behavior.