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PubMed1999

The Gas7 gene encodes two protein isoforms differentially expressed within the brain.

Gas7, a growth arrest-specific gene first isolated from serum-starved NIH3T3 cells, is expressed abundantly in the brain and is essential for the outgrowth of neurites from cultured cerebellar neurons. Here, we report the existence of a Gas7-related cDNA, designated Gas7-cb, isolated from the mouse cerebellum, and we report the finding that Gas7-cb transcripts and protein are expressed at different locations than those of Gas7. Gas7-cb cDNA differs from the Gas7 cDNA only in the 5' region. Its encoded protein shares the same 320 amino acids in its C-terminus with those of Gas7. Analyses of the RNA and protein expression of Gas7-cb and Gas7 by RNase protection assay and Western blot indicated that while Gas7 expression is predominant in the cerebrum and in growth-arrested NIH3T3 fibroblasts, Gas7-cb expression is predominant in the cerebellum. Characterization of Gas7 and Gas7-cb RNAs and of the genomic structure of murine Gas7 cloned in a bacterial artificial chromosome indicated that the Gas7 gene spans more than 60 kb and consists of at least 15 exons. The 5'-terminus of Gas7-cb is located at exon 6a, which is absent in Gas7 transcripts but is retained in its entirety in Gas7-cb transcripts, resulting in the presence of a unique 20-amino-acid sequence at the N-terminus of the Gas7-cb protein. Our results show that the Gas7 gene encodes two Gas7 isoforms, Gas7 and Gas7-cb, whose expression is differentially regulated within mouse brain.

3T3 Cells↗
PubMed1997

Direct identification of differentially expressed genes by cycle sequencing and cycle labelling using the differential display PCR primers.

Differential display PCR (DD-PCR) is an mRNA fingerprinting technique to identify differentially expressed genes by comparative display of arbitrarily amplified cDNA subsets. This attractively simple screening method was, however, followed by a labour intensive multistep identification procedure for DD-PCR products. In this report we demonstrate for the mouse mast cell protease 2 (MMCP-2) and the cytotoxic T-lymphocyte associated gene transcript CTLA-1 a streamlined approach by (i) direct cycle sequencing with the upstream differential display (DD) primer, followed by (ii) the PCR based generation of an antisense northern probe with the downstream anchor primer.

Animals↗
PubMed1997

Differential expression of elongation factor-2, alpha4 phosphoprotein and Cdc5-like protein in prolactin-dependent/independent rat lymphoid cells.

The differential display of mRNAs technique was used to identify genes which are differentially expressed in the prolactin (PRL)-dependent Nb2-11C and PRL-independent Nb2-Sp rat lymphoma cell lines. The technique was validated by the differential display of the proto-oncogene c-myc in PRL-treated, but not in untreated, Nb2-11C cells. Nine DNA bands, isolated from the 6% display gels and confirmed by reverse Northerns to be differentially expressed, were cloned and gave rise to ten unique clones. DNA sequencing showed that these clones had limited homologies to known genes or uncharacterized expressed sequence tags. Using one of these clones (6ac1.12) as a probe in Northern analysis, a transcript of approximately 4 kb was detected which was elevated in PRL-treated Nb2-11C cells and in mid-log phase growing Nb2-Sp cells. Similar to c-myc, expression of the 4 kb transcript increased in Nb2-11C cells given PRL for 3 h (+/- the phorbol ester TPA) but not in cells given TPA alone. The 4 kb transcript also increased with increasing Nb2-11C cell densities. By screening an Nb2-Sp cDNA library with 6ac1.12 as probe, three unique genes were isolated and identified as elongation factor 2 (EF-2), alpha4 phosphoprotein and a Cdc5-like protein. Each of the three genes were PRL responsive in Nb2-11C cells and expressed constitutively in Nb2-Sp cells. The expression of EF-2 or alpha4, but not the Cdc-like protein, was dependent on cell densities. EF-2 regulates protein synthesis while the alpha4 and Cdc5-like phosphoproteins have been implicated in IgG receptor-mediated and mitogen-activated signaling, respectively. The identification that these genes are PRL-responsive and/or differentially expressed in the Nb2-11C and Nb2-Sp cell lines may permit insights into the molecular changes that are involved in regulating the transition to growth factor independence in lymphoid tumors.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Differential expression of Vibrio vulnificus elastase gene in a growth phase-dependent manner by two different types of promoters.

Elastase activity of Vibrio vulnificus was highly dependent on growth phase, reached a maximum during the stationary phase, and was regulated at the level of transcription. The stationary phase production of elastase in crp or rpoS mutants, which were constructed by allelic exchanges, decreased about 3- and 10-fold, respectively. However, the promoter activity of vvpE encoding elastase was unaffected by those mutations in the log phase when analyzed using a vvpE-lux fusion. A primer extension analysis revealed that the transcription of vvpE begins at two different sites, consisting of putative promoter L (PL) and promoter S (PS). The PL activity was constitutive through the log and stationary phases, lower than the PS activity, and unaffected by the crp or rpoS mutations. The transcription of PS, induced only in the stationary phase, was dependent on RpoS. The mutation in crp reduced the activity of PS; however, the additional inactivation of crp did not influence the PS activity in the rpoS mutant, indicating that CRP exerted its effects through PS requiring RpoS. These results demonstrate that vvpE expression is differentially directed by PL and PS depending on the growth phase and elevated by RpoS and CRP in the stationary phase.

Alleles↗
PubMed2001

Genes differentially expressed in the kindled mouse brain.

Kindling involves long-term changes in brain excitability and is considered a model of epilepsy and neuroplasticity. Differentially expressed genes in the kindled mouse brain were screened using an reverse transcription-polymerase chain reaction (RT-PCR) differential display (DD) method. C3H male mice were kindled with 40 stimuli in the hippocampus at 5-min intervals. Hippocampal RNA was isolated for DD from mice at 0.5 h, 1 day, 1 week, and 1 month after kindling and from sham-operated controls. About 30,000 bands were screened and of these, 50 were displayed differentially. Northern blot analysis confirmed that 26 of the 50 bands were differentially expressed following rapid kindling. Further sequence analysis revealed that 14 of the genes were previously identified and 12 were novel. The novel genes are referred to as King (1-12) genes because of their association with kindling. According to their temporal and quantitative pattern of expression in forebrain, the 26 genes were grouped into five types. Expression of five of the DD genes, one from each expression type, was further analyzed in hippocampus, forebrain, brainstem, and cerebellum of the kindled mice. Differential expression of these genes was observed in hippocampus and forebrain, but not in brainstem or cerebellum. Only one gene, a regulator of G-protein signaling 4 (RGS4), showed prolonged changes in expression in response to kindling. Our results show that rapid kindling produces spatial and temporal changes in gene expression that may influence kindling-associated neuroplasticity.

Animals↗
PubMed1999

Exhaustive mining of EST libraries for genes differentially expressed in normal and tumour tissues.

A four-step procedure for the efficient and systematic mining of whole EST libraries for differentially expressed genes is presented. After eliminating redundant entries from the EST library under investigation (step 1), contigs of maximal length are built upon each remaining EST using about 4 000 000 public and proprietary ESTs (step 2). These putative genes are compared against a database comprising ESTs from 16 different tissues (both normal and tumour affected) to determine whether or not they are differentially expressed (step 3; electronic northern). Fisher's exact test is used to assess the significance of differential expression. In step 4, an attempt is made to characterise the contigs obtained in the assembly through database comparison. A case study of the CGAP library NCI_CGAP_Br1.1, a library made from three (well, moderately, and poorly differentiated) invasive ductal breast tumours (2126 ESTs in total) was carried out. Of the maximal contigs, 139 were found to be significantly (alpha = 0.05) over-expressed in breast tumour tissue, while 13 appeared to be down-regulated.

Animals↗
PubMed2003

Statistical methods for ranking differentially expressed genes.

In the analysis of microarray data the identification of differential expression is paramount. Here I outline a method for finding an optimal test statistic with which to rank genes with respect to differential expression. Tests of the method show that it allows generation of top gene lists that give few false positives and few false negatives. Estimation of the false-negative as well as the false-positive rate lies at the heart of the method.

Computational Biology↗
PubMed2004

Identification of differentially expressed genes of primary spermatocyte against round spermatid isolated from human testis using the laser capture microdissection technique.

The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed during differentiation and development. In the present study, over 10,000 primary spermatocytes and round spermatid cells were successfully isolated by LCM. Using the cDNAs from primary spermatocytes and round spermatids, SSH cDNAs library of primary spermatocyte-specific was constructed. The average insert size of the cDNA isolated from 75 randomly picked white clones was 500 bp, ranging from 250 bp to 1.7 kb. Using the dot-blot method, a total of 421 clones were examined, resulting in the identification of 390 positive clones emitting strong signals. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. Genes encoding cytochrome c oxidase II and the rescue factor-humanin were most frequently expressed in primary spermatocytes, suggesting their roles involved in meiosis.

Cloning, Molecular↗
PubMed2006

Differentially expressed genes in the prostate cancer cell line LNCaP after exposure to androgen and anti-androgen.

Androgens play an important role in growth and maintenance of prostate cells. The actions of androgens are mediated by the androgen receptor (AR), a transcription factor member of the super-family of nuclear hormone receptors. Androgen regulated genes (ARGs) are potential markers for early diagnosis and treatment of prostate cancer patients. In the present study, we used DDRT-PCR (differential display reverse transcriptase polymerase chain reaction) technique in order to investigate differentially expressed genes in the prostate cancer cell line LNCaP after treatment with dihydrotestosterone and bicalutamide for 6, 24, and 48 hours. Fifty-five differentially expressed fragments were isolated, cloned, and sequenced. Sequencing analysis of these fragments revealed 56 different transcripts that showed homology to transcription factors, cell cycle regulators, metabolic enzymes, and hypothetical proteins. Among the differentially expressed genes, SPA17 and DDEF2 were further validated using quantitative real time RT-PCR (qPCR) in a series of 25 prostate tumor samples. The DDEF2 gene is involved in adhesion and cell migration of monocytes, and the SPA17 gene might be involved in cellular signal transduction. The transcripts of both, SPA17 and DDEF2 genes, showed altered pattern of expression in the group of prostate tumors analyzed by qPCR. The differentially expressed genes identified in this study might provide new insights into the androgen signaling pathways in prostate cells.

Aged↗
PubMed2001

Differential expression of neuroleukin in osseous tissues and its involvement in mineralization during osteoblast differentiation.

Osteoblast differentiation is a multistep process that involves critical spatial and temporal regulation of cellular processes marked by the presence of a large number of differentially expressed molecules. To identify key functional molecules, we used differential messenger RNA (mRNA) display and compared RNA populations isolated from the defined transition phases (proliferation, matrix formation, and mineralization) of the MC3T3-E1 osteoblast-like cell line. Using this approach, a complementary DNA (cDNA) fragment was isolated and identified as neuroleukin (NLK), a multifunctional cytokine also known as autocrine motility factor (AMF), phosphoglucose isomerase (PGI; phosphohexose isomerase [PHI]), and maturation factor (MF). Northern analysis showed NLK temporal expression during MC3T3-E1 cell differentiation with a 3.5-fold increase during matrix formation and mineralization. Immunocytochemical studies revealed the presence of NLK in MC3T3-E1 cells as well as in the surrounding matrix, consistent with a secreted molecule. In contrast, the NLK receptor protein was detected primarily on the cell membrane. In subsequent studies, a high level of NLK expression was identified in osteoblasts and superficial articular chondrocytes in bone of 1-, 4-, and 8-month-old normal mice, as well as in fibroblasts, proliferating chondrocytes, and osteoblasts within a fracture callus. However, NLK was not evident in hypertrophic chondrocytes or osteocytes. In addition, treatment of MC3T3 cells with 6-phosphogluconic acid (6PGA; a NLK inhibitor) resulted in diminishing alkaline phosphatase (ALP) activity and mineralization in MC3T3-E1 cells, especially during the matrix formation stage of differentiating cells. Taken together, these data show specific expression of NLK in discrete populations of bone and cartilage cells and suggest a possible role for this secreted protein in bone development and regeneration.

3T3 Cells↗
PubMed1998

Identification of genes differentially expressed by hypoxia in hepatocellular carcinoma cells.

In order to identify genes differentially expressed under hypoxia (1% O2, 5% CO2, balance N2), we performed mRNA differential display analysis using total RNA extracted from hypoxic and normoxic HepG2, human hepatocellular carcinoma (HCC) cells. Of the differentially expressed genes by hypoxia, some of cDNA fragments were cloned and sequenced. The expression patterns of these clones by hypoxia were confirmed by Northern blot analysis and the quantitative RT-PCR. Down-regulated genes by hypoxia have homology to cDNA sequences encoding cytochrome oxidase subunit II and ADP/ATP translocase, respectively. Up-regulated gene by hypoxia was identified as Homo sapiens oscillin. Moreover, novel genes induced by hypoxia represent partial sequences of cDNAs that have not been reported or functionally identified. Up- or down-regulated expression of these genes in response to hypoxia may contribute to human hepatocarcinogenesis.

Amino Acid Sequence↗
PubMed1999

Differential expression of genes for cyclin-dependent protein kinases in rice plants.

Cyclin-dependent protein kinases (CDKs) play key roles in regulating the eukaryotic cell cycle. We have analyzed the expression of four rice (Oryza sativa) CDK genes, cdc2Os1, cdc2Os2, cdc2Os3, and R2, by in situ hybridization of sections of root apices. Transcripts of cdc2Os1, cdc2Os2, and R2 were detected uniformly in the dividing region of the root apex. cdc2Os1 and cdc2Os2 were also expressed in differentiated cells such as those in the sclerenchyma, pericycle, and parenchyma of the central cylinder. By contrast, signals corresponding to transcripts of cdc2Os3 were distributed only in patches in the dividing region. Counterstaining of sections with 4', 6-diamidino-2-phenylindole and double-target in situ hybridization with a probe for histone H4 transcripts revealed that cdc2Os3 transcripts were abundant from the G2 to the M phase, but were less abundant or absent during the S phase. The levels of the Cdc2Os3 protein and its associated histone H1-kinase activity were reduced by treatment of cultured cells with hydroxyurea, which blocks cycling cells at the onset of the S phase. Our results suggest that domains other than the conserved amino acid sequence (the PSTAIRE motif) have important roles in the function of non-PSTAIRE CDKs in distinct cell-cycle phases.

Amino Acid Sequence↗
PubMed2006

Proteomics identification of differentially expressed proteins associated with pollen germination and tube growth reveals characteristics of germinated Oryza sativa pollen.

Mature pollen from most plant species is metabolically quiescent; however, after pollination, it germinates quickly and gives rise to a pollen tube to transport sperms into the embryo sac. Because methods for collecting a large amount of in vitro germinated pollen grains for transcriptomics and proteomics studies from model plants of Arabidopsis and rice are not available, molecular information about the germination developmental process is lacking. Here we describe a method for obtaining a large quantity of in vitro germinating rice pollen for proteomics study. Two-dimensional electrophoresis of approximately 2300 protein spots revealed 186 that were differentially expressed in mature and germinated pollen. Most showed a changed level of expression, and only 66 appeared to be specific to developmental stages. Furthermore 160 differentially expressed protein spots were identified on mass spectrometry to match 120 diverse protein species. These proteins involve different cellular and metabolic processes with obvious functional skew toward wall metabolism, protein synthesis and degradation, cytoskeleton dynamics, and carbohydrate/energy metabolism. Wall metabolism-related proteins are prominently featured in the differentially expressed proteins and the pollen proteome as compared with rice sporophytic proteomes. Our study also revealed multiple isoforms and differential expression patterns between isoforms of a protein. These results provide novel insights into pollen function specialization.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2001

Identification of differentially expressed genes in normal mucosa, adenoma and adenocarcinoma of colon by SSH.

AIM: To construct subtracted cDNA libraries and further identify differentially expressed genes that are related to the development of colorectal carcinoma(CRC). METHODS: Suppression subtractive hybridization(SSH) was done on cDNAs of normal mucosa, adenoma and adenocarcinoma tissues from the same patient. Three subtracted cDNA libraries were constructed and then hybridized with forward and backward subtracted probes for differential screening. Positive clones from each subtracted cDNA library were selected for sequencing and BLAST analysis. Finally, virtual Northern Blot confirmed such differential expression. RESULTS: By this way, there were about 3-4 X 10(2) clones identified in each subtracted cDNA library, in which about 85% positive clones were differentially screened. Sequencing and BLAST homology search revealed some clones containing sequences of known gene fragments and several possibly novel genes showing few or no sequence homologies with any known sequences in the database. CONCLUSION: All results confirmed the effectiveness and sensitivity of SSH. The differentially expressed genes during the development of CRC can be used to shed light on the pathogenesis of CRC and be useful genetic markers for early diagnosis and therapy.

Adenocarcinoma↗
PubMed2003

Differential expression and localization of the phosphorylated and nonphosphorylated neurofilaments during the early postnatal development of rat hippocampus.

Neurofilament (NF) proteins are expressed in most mature neurons in the central nervous system. Although they play a crucial role in neuronal growth, organization, shape, and plasticity, their expression pattern and cellular distribution in the developing hippocampus remain unknown. In the present study, we have used Western blotting and immunocytochemistry to study the low- (NF-L), medium- (NF-M), and high- (NF-H) molecular-weight NF proteins; phosphorylated epitopes of NF-M and NF-H; and a nonphosphorylated epitope of NF-H in the early postnatal (through P1-P21) development of the rat hippocampus. During the first postnatal week, NF-M was the most abundantly expressed NF, followed by NF-L, whereas the expression of NF-H was very low. Through P7-P14, the expression of NF-H increased dramatically and later began to plateau, as also occurred in the expression of NF-M and NF-L. At P1, no NF-M immunopositive cell bodies were detected, but cell processes in the CA1-CA3 fields were faintly immunopositive for NF-M and for the phosphorylated epitopes of NF-M and NF-H. At P7, CA3 pyramidal neurons were strongly immunopositive for NF-L and NF-H, but not for NF-M. The axons of granule cells, the mossy fibers (MFs), were NF-L and NF-M positive through P7-P21 but were NF-H immunonegative at all ages. Although they stained strongly for the phosphorylated NF-M and NF-H at P7, the staining intensity sharply decreased at P14 and remained so at P21. The cell bodies of CA1 pyramidal neurons and granule cells remained immunonegative against all five antibodies in all age groups. Our results show a different time course in the expression and differential cell type and cellular localization of the NF proteins in the developing hippocampus. These developmental changes could be of importance in determining the reactivity of hippocampal neurons in pathological conditions in the immature hippocampus.

Aging↗
PubMed1990

Differential expression of excitatory amino acid receptor subtypes in cultured cerebellar neurons.

Using neurotoxicity and inositol phosphate release as criteria for receptor expression, we report the differential expression of excitatory amino acid receptor subtypes in cerebellar granule cells grown in serum-free media containing either high (25 mM) or low (5 mM) KCl. NMDA receptors are expressed in neurons grown in high, but not low, KCl. In contrast, ionotropic quisqualate receptors are expressed in neurons grown in low KCl, but not in those grown in high KCl. Addition of NMDA to cultures containing low KCl appears to mimic high KCl conditions: NMDA receptors are expressed, but ionotropic quisqualate receptors are not. Glutamate and kainate are toxic to cells grown in either condition.

Animals↗
PubMed2006

Differential expression of HPA axis homolog in the skin.

Human skin expresses elements of the hypothalamo-pituitary-adrenal (HPA) axis including pro-opiomelanocortin (POMC), corticotropin releasing hormone (CRH), the CRH receptor-1 (CRH-R1), key enzymes of corticosteroid synthesis and synthesizes glucocorticoids. Expression of these elements is organized in functional, cell type-specific regulatory loops, which imitate the signaling hierarchy of the HPA axis. In melanocytes and fibroblasts CRH-induced CRH-R1 stimulation upregulates POMC expression and production of ACTH through activation of cAMP dependent pathway(s). Melanocytes respond with enhanced production of cortisol and corticosterone, which is dependent on POMC activity. Fibroblasts respond to CRH and ACTH with enhanced production of corticosterone, but not cortisol, which is produced constitutively. Organ-cultured human scalp hair follicles also show a fully functional HPA axis equivalent, including cortisol synthesis and secretion and negative feedback regulation by cortisol on CRH expression. Thus, differential, CRH-driven responses of defined cutaneous cell populations reproduce key features of the central HPA axis at the tissue/single cell levels.

Animals↗