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Results for “Expression analysis”

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PubMed1999

Functional characterization and expression analysis of the amino acid permease RcAAP3 from castor bean.

A polymerase chain reaction-based library screening procedure was used to isolate RcAAP3, an amino acid permease cDNA from castor bean (Ricinus communis). RcAAP3 is 1.7 kb in length, with an open reading frame that encodes a protein with a calculated molecular mass of 51 kD. Hydropathy analysis indicates that the RcAAP3 protein is highly hydrophobic in nature with nine to 11 putative transmembrane domains. RcAAP3-mediated uptake of citrulline in a yeast transport mutant showed saturable kinetics with a K(m) of 0.4 mM. Transport was higher at acidic pH and was inhibited by the protonophore carbonylcyanide-m-chlorophenylhydrazone, suggesting a proton-coupled transport mechanism. Citrulline uptake was strongly inhibited (72%) by the permeable sulfydryl reagent N-ethylmaleimide, but showed lower sensitivity (30% inhibition) to the nonpermeable reagent p-chloromercuribenzenesulfonic acid. Diethylpyrocarbonate, a histidine modifier, inhibited citrulline uptake by 80%. A range of amino acids inhibited citrulline uptake, suggesting that RcAAP3 may be a broad substrate permease that can transport neutral and basic amino acids with a lower affinity for acidic amino acids. Northern analysis indicated that RcAAP3 is widely expressed in source and sink tissues of castor bean, and that the pattern of expression is distinct from RcAAP1 and RcAAP2.

Amino Acid Sequence↗

PubMed2005

Identification and expression analysis of regulatory genes induced during conidiation in Exserohilum turcicum.

Light influences numerous developmental and biochemical processes in fungi. The objectives of this research were to characterize the influence of light on growth and conidiation and associated gene expression in the plant pathogenic ascomycete, Exserohilum turcicum. We found that vegetative growth was more extensive in light/dark cycles than in constant light or darkness as measured by analysis of ergosterol content and genomic DNA. Cultures grown under continuous white light or blue light (approximately 465-480 nm) were developmentally arrested after the formation of conidiophores, whereas those grown in continuous darkness or a light/dark cycle produced mature conidia. Incubation of conidiophore-producing cultures in darkness for a minimum of 2 h was necessary and sufficient to initiate synchronous conidiation. To identify genes that are expressed during dark-induced conidiation, we constructed subtractive cDNA libraries from cultures grown under conidiation-permissive and -repressive conditions. From 816 sequenced EST clones in the conidiation-permissive and 310 in the repressive libraries, 12 putative regulatory genes were chosen for expression analysis by quantitative real-time PCR. The majority of those genes reached maximum expression by 2 h after initiation of the dark period and then declined to initial levels by 4-24 h in darkness. Expression of two dark-induced genes remained elevated after 24 h in darkness but was reset to initial levels if cultures were returned to light. This study revealed several genes whose expression increased rapidly after dark induction of conidiation, suggesting that they encode regulators of asexual development in E. turcicum.

Ascomycota↗

PubMed2004

Expression analysis of members of the neuronal calcium sensor protein family: combining bioinformatics and Western blot analysis.

We have used in silico mining of public databases (NCBI UniGene and NCI SAGE Anatomic Viewer) as a tool to obtain the tissue distribution pattern of three members of the neuronal calcium sensor protein family, namely VILIP-1, hippocalcin, and NCS-1 in humans. The theoretical human mRNA expression profile of the calcium sensor proteins derived from expressed sequence tag (EST) and serial analysis of gene expression (SAGE) data was compared with expression data from human tissues obtained by Western blot analysis. Since the EST databank searches do not yet give comparable results for rat which is often used as model animal, we have also analyzed the protein expression in rat tissues. Similar to the human expression profile in rat tissues calcium sensor proteins are mainly detected in the nervous system, but the data consistently implicated the additional expression in peripheral tissues with remarkable differences between the calcium sensor proteins.

Animals↗

PubMed2002

Molecular cloning and expression analysis of dystroglycan during Xenopus laevis embryogenesis.

Dystroglycan is a transmembrane receptor protein that provides a structural linkage between extracellular matrix components and cytoskeletal proteins. It was originally characterized as a member of dystrophin associated protein complex in muscle but, unlike other proteins of this complex, mutations in the dystroglycan gene have not been implicated as a cause of muscular dystrophies. Indeed, dystroglycan is an essential gene, expressed early in development that, if removed in knockout mice, provokes lethal defects before the onset of myogenesis. Dystroglycan is synthesized as a precursor propeptide that is post-translationally cleaved and glycosylated to yield alpha and beta subunits. We have cloned and characterized a cDNA clone, containing the complete coding region of the dystroglycan precursor, from a Xenopus laevis cDNA library. We have performed a spatial and temporal analysis of its expression in X. laevis embryos, using whole-mount in situ hybridization and reverse transcription-polymerase chain reaction analysis. Early expression of dystroglycan in a variety of tissues of different embryological derivation suggests a crucial role in morphogenetic events, especially during central nervous system differentiation.

Amino Acid Sequence↗

PubMed2002

Molecular cloning and expression analysis of dystroglycan during Xenopus laevis embryogenesis.

Animals↗

PubMed1998

Mutation and expression analysis of the p27/kip1 gene in corticotrophin-secreting tumours.

The molecular mechanisms leading to Cushing's disease are unclear. Inhibitors of cyclin-cyclin dependent kinase (CDK) complexes are regulators of the cell cycle and may function as tumour suppressor genes, many of which have been involved in the pathogenesis of several human malignancies. A member of this family, the p27/kip1 gene, maps to chromosome 12p13 and encodes an inhibitor of several cyclin-CDK complexes; these control the progression of the cell cycle from G1 to S-phase. Complete lack of p27/kip1 function, as occurs in the p27/kip1 'knockout' mouse, produces a complex phenotype associated with the development of pituitary tumours, specifically those of the intermediate lobe corticotrophs. We therefore investigated whether structural and functional abnormalities of the p27/kip1 gene and loss at the chromosome 12p13 region were present in human corticotrophin (ACTH)-secreting pituitary tumours. We studied 21 pituitary tumours, of which 20 were ACTH-secreting (two of these had biochemical and histological features of 'intermediate-lobe' tumours and one was malignant) while the remaining tumour was a prolactinoma; three ectopic secretors of ACTH (two bronchial and one thymic carcinoid); and a non-secretory thymic carcinoid. The whole coding region of the p27/ kip1 gene was screened for mutations by PCR-SSCP analysis and/or direct sequencing, while tumour mRNA expression was analysed by means of a semi-quantitative duplex PCR. Three polymorphic microsatellite markers of the 12p13 region were used to assess loss of heterozygosity (LOH) in 12 samples. Finally, tumour p27/kip1 protein expression was assessed by immunohistochemistry using a monoclonal antibody in 12 samples suitable for analysis. No sequence abnormalities were found in any of the samples other than a previously-described polymorphism. No LOH was observed in the tumours analysed. p27/kip1 mRNA expression was similar in tumour samples in comparison with normal pituitaries. Seven of the eight corticotroph tumours analysed by immunohistochemistry stained positive for p27/kip1, including the intermediate lobe. The only malignant pituitary tumour in the original series showed an absence of staining for p27/kip1. In addition, the three carcinoid tumours studied were negative on immunohistochemistry. Of a further three malignant pituitary tumours assessed, two (including a prolactinoma) were essentially negative, while the third was moderately positive. We conclude that mutations of the p27/kip1 gene, deletions of the 12p13 area or changes in expression, are not a general feature of corticotroph tumours, even those with intermediate lobe characteristics. However, other mechanisms of p27/kip1 inactivation, such as an abnormality at the post-translational level, may be related to more aggressive histological subtypes of ACTH-secreting and possibly other pituitary tumours.

Adrenocorticotropic Hormone↗

PubMed2026

DiaReport: reproducible workflow for differential expression analysis and interactive reporting in DIA-based proteomics.

MOTIVATION: Data-independent acquisition (DIA) has become the preferred data acquisition method for mass spectrometry-based proteomics, yet, reproducible workflows for differential expression (DE) analysis and results reporting remain limited. We present DiaReport, an R package that performs precursor- and protein-level DE analysis from DIA-NN output using MSqRob and QFeatures, while generating high-quality, interactive HTML reports through Quarto. DiaReport integrates precursor data, filtering of missing values, normalization, protein summarization and statistical modeling within a single function, supporting both simple pairwise as well as complex experimental designs. The package provides structured outputs and configuration files to ensure computational reproducibility across different studies. To accommodate diverse research needs, DiaReport includes multiple reporting templates tailored to different proteomic applications. Applying DiaReport to an extracellular vesicle (EV) proteomics dataset demonstrates its ability to efficiently analyze DIA data and provide rapid insights into sample quality and protein level differences. AVAILABILITY: DiaReport is an open-source R package available at https://github.com/Gevaert-Lab/diareport (DOI: 10.5281/zenodo.20120604). The package is platform-independent and distributed under the MIT license. Reports are generated using Quarto and require only standard R dependencies. Detailed documentation, installation guides and usage vignettes are provided within the repository. The interactive HTML reports discussed in this study, including the UPS2 benchmark and EV case study, are archived on Zenodo (10.5281/zenodo.20122506 and 10.5281/zenodo.20123378).

Proteomics↗

PubMed2006

Gene expression analysis in mice liver on hepatocarcinogenesis by flumequine.

mRNA expression profiles in the liver from mice treated with flumequine (FL) were analyzed in order to elucidate the mechanism of its tumor-promoting effect. The liver from a C3H/He mouse that received a diet containing 4,000 ppm of FL for 4 weeks was examined by cDNA microarray in comparison with an untreated mouse. Furthermore, to obtain a more comprehensive sequence, time-course changes in selected genes were determined by real-time RT-PCR. Microarray analysis revealed 15 upregulated and 9 downregulated genes in an FL-treated mouse. The upregulated genes included signal transducers and cell cycle regulators. In addition, the levels of stress response genes, particularly glutathione S-transferase (GST) alpha and GSTmu, were very high, indicating the generation of oxidative stress. On the other hand, the downregulated genes included phase I metabolic enzymes, such as cytochrome P450 (CYPs) enzymes, and apoptosis-associated proteins. These changes were confirmed by quantitative RT-PCR and were generally consistent with each other. Time-course observations revealed consistent results, particularly with regard to GSTalpha, GSTmu, ERK5, and CYP2E1. In addition, the expression of 8-oxoguanine DNA glycosylase 1 (OGG1) was increased in a time-dependent manner. These results suggest the possibility that responses against oxidative stress may play a major role in hepatocarcinogenesis by FL in mice.

Animals↗

PubMed2003

[Cloning and expression analysis of lung carcinoma related gene HLCDG1].

BACKGROUND & OBJECTIVE: High frequent loss of hetero- zygosity (LOH) of 3p, 5q, 6q, 9p, 10q, 11p, 13q, 17p, and 19p in lung carcinoma was detected by comparative genomic hybridization (CGH) and genomic-wide scan for analysis of genetic alteration with microsatellite allelotying. It was indicated that there might be some other unknown tumor susceptible genes or suppressor genes likely to be involved in lung carcinoma development and progression. The aim of this study was to clone the full-length cDNA of LXDD1,an expressed sequence tag(EST) isolated by mRNA differential display, which is significantly down-regulated in lung carcinoma and represents a novel gene. METHODS: Differential expression of LXDD1 in lung carcinoma was confirmed by Northern blot analysis, the expression of the LXDD1 in human normal tissues and the size of the transcription of the LXDD1-representative gene were also determined using MTN (Multiple Tissues Northern Blots). The putative full-length cDNA of the EST-representative gene was cloned and analyzed by bioinformatics. In addition, differential reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of the novel gene in various cancer cell lines, primary lung carcinomas, and matched normal lung tissues. RESULTS: The full length cDNA with no homology to any reported genes in the database of GenBank was successfully cloned and named HLCDG1 (Human lung carcinoma deleted gene 1, GenBank accession number AF447582). A transmembrane protein with 166 amino acids was deduced to come from the open reading frame of the 3113 bp full-length cDNA, HLCDG1 gene was confirmed to be located at chromosome band 5q33 by alignment of electric polymerase chain reaction (e-PCR). CONCLUSION: HLCDG1 is a novel gene down-regulated in lung carcinoma, which may be involved in the development of lung carcinoma.

Base Sequence↗

PubMed2006

Characterization and expression analysis of the cytochrome bd oxidase operon from Desulfovibrio gigas.

Although classified as anaerobic, Desulfovibrio gigas contains a functional canonical membrane respiratory chain, including a cytochrome bd quinol oxidase as its terminal element. In the present study, we report the identification of the operon cydAB encoding the two subunits of cytochrome bd from this bacterium. Two hypothetical promoter regions and sequences resembling transcriptional regulators-binding sites have been identified. Amino acid sequence analysis revealed a high similarity to cytochrome bd from other organisms, presenting the conserved residues typical from these proteins. Reverse transcription polymerase chain reaction (RT-PCR) and Northern blot analysis confirmed the operon transcription. Gene expression was assessed by real-time RT-PCR in cells grown in different media and under exposure to oxygen and nitric oxide. mRNA levels were slightly enhanced in the presence of 150 microM: NO. However, in the presence of 10 microM: NO, a decrease was observed of the steady-state population of cydAB mRNA. No considerable effect was observed in the presence of fumarate/sulfate medium, 60 microM: O2 or 10 microM: NO.

Amino Acid Sequence↗

PubMed2000

Molecular cloning, expression analysis, and chromosome mapping of WDR6, a novel human WD-repeat gene.

The WD-repeat proteins are found in all eukaryotes and play an important role in the regulation of a wide variety of cellular functions such as signal transduction, transcription, and proliferation. Here we report on the cloning and characterization of a novel human WD-repeat gene, WDR6, which encodes a protein of 1121 amino acids and contains 11 WD-repeat units. WDR6 is unique since its 11 WD repeats are clustered into two distinct groups separated by a putative transmembrane domain. The WDR6 gene was mapped to chromosome 15q21 by fluorescence in situ hybridization. Northern analysis demonstrated that WDR6 is ubiquitously expressed in human adult and fetal tissues. WDR6 is not homologous to any previously identified human WD-repeat genes including WDR1 through WDR5. However, it was found to have significant sequence similarity with Arabidopsis thaliana hypothetical protein T7B11.12, yeast putative elongation factor G, and probable membrane protein YPL183c. All of them have been defined as WD-repeat proteins. Therefore, WDR6 is a novel protein and probably belongs to a highly conserved subfamily of WD-repeat proteins in which T7B11.12 and YPL183c are its distantly related members.

Amino Acid Sequence↗

PubMed1997

Cloning, sequence, and developmental expression analysis of C4-2, a potential brain tumor-suppressor gene.

BACKGROUND: Previously, we reported the isolation of C4-2 as a potential tumor suppressor gene in human brain tumors. To understand the function of this gene, we investigated its molecular characterization and expression during development. METHODS: Human fetal brain library screening and 5'RACE-PCR method was used to isolate the full-length cDNA. The coding region of C4-2 was used for in situ hybridization to study its expression during development. RESULTS: We report here the complete sequence of this gene. Sequence analysis indicated that C4-2 has a 94% sequence identity to a family of cAMP-regulated phosphoproteins (ARPP-16/19) in the coding region. C4-2 has a 3.1 Kb long 3'UTR with variable identity to ARPP-16 and ARPP-19. Northern blot analysis indicated that C4-2 is expressed at high levels in normal brain compared to other tissues. Zoo blot analysis demonstrated that the coding region of C4-2 is highly conserved among different animals. In situ hybridization using C4-2 coding region demonstrated that it follows a unique expression pattern during mouse brain development. High level of C4-2 expression was also observed in the spinal cord and somites of the developing embryo. CONCLUSION: Expression analysis during brain development strongly suggests that this family of proteins may play an important role not only in normal functioning of the brain, but also during brain development.

Amino Acid Sequence↗

PubMed2004

Expression analysis of a chicory fructan 1-exohydrolase gene reveals complex regulation by cold.

The gene for a recently identified cDNA, 1-FEH IIa, encoding a fructan 1-exohydrolase was isolated and cloned from Cichorium intybus and a 1149 bp promoter fragment was characterized. An analysis of the genomic 1-FEH IIa sequence indicated that the gene (FEHIIa) consists of six introns and seven exons, which is similar to plant invertase genes. Like invertase genes, FEHIIa also contains the 9 nt mini-exon encoding the tripeptide DPN. A database search for cis-acting response elements within its promoter identified multiple elements that appear to have relevance to cold-induced expression of the gene in field-grown roots. Promoter analysis by transient expression assay demonstrated that the FEHIIa gene promoter is highly expressed in etiolated Cichorium leaves and cold-stored roots, which correlated well with the high level expression detected by RNA blot analysis. Cold also enhanced FEHIIa reporter gene expression in green leaves, however, the reporter gene activity was much lower compared with similar induction experiments in etiolated leaves. Promoter deletion analysis demonstrated the presence of potential cold-responsive ABRE and/or CRT/DRE elements in the -22 to -172 region, while regions -933 to -717 and -493 to -278 contain elements that can down-regulate expression at the conditions used. Characterization of the FEHIIa promoter may provide tools to study cold-induced expression and to increase freezing tolerance in agricultural crops.

Base Sequence↗

PubMed2005

Differentiating mechanisms of toxicity using global gene expression analysis in Saccharomyces cerevisiae.

Genotoxic stress triggers a variety of biological responses including the transcriptional activation of genes regulating DNA repair, cell survival and cell death. Genomic approaches, which monitor gene expressions across large numbers of genes, can serve as a powerful tool for exploring mechanisms of toxicity. Here, using five different agents, we investigated whether the analysis of genome-wide expression profiles in Saccharomyces cerevisiae could provide insights into mechanisms of genotoxicity versus cytotoxicity. To differentiate the genotoxic stress-associated expression signatures from that of a general cytotoxic stress, we compared gene expression profiles following the treatment with DNA-reactive (cisplatin, MMS, bleomycin) and DNA non-reactive (ethanol and sodium chloride) compounds. Although each of the tested chemicals produced a distinct gene expression profile, we were able to identify a gene expression signature consisting of a relatively small number of biologically relevant genes capable of differentiating genotoxic and cytotoxic stress. The gene set includes such upregulated genes as HUG1, ECM4 and previously uncharacterized gene, YLR297W in the genotoxic and GAP1, CGR1 in the cytotoxic group. Our results indicate the potential of gene expression profile analysis for elucidating mechanism of action of genotoxic agents.

Alkylating Agents↗

PubMed1990

cDNA isolation, expression analysis, and chromosomal localization of two human zinc finger genes.

On the basis of sequence similarity in the repeated zinc finger domain, we have identified and characterized two human cDNA clones (ZNF7 and ZNF8), both encoding proteins containing potential finger-like nucleic acid binding motifs. Northern blot analysis indicates that both genes are expressed as multiple transcripts and they are ubiquitously present in many human cell lines of different embryological derivation. Moreover, their expression is modulated during in vitro induced terminal differentiation of human myeloid cell line HL-60. By in situ hybridization experiments, we have localized the ZNF7 gene to chromosome 8 (region q24) and the ZNF8 gene to the terminal band of the long arm of chromosome 20 (20q13).

Amino Acid Sequence↗

PubMed2006

Cloning and expression analysis of androgen receptor gene in chicken embryogenesis.

We cloned a full-length androgen receptor (AR) cDNA from chicken (Gallus gallus) gonads. The cDNA sequence has an open reading frame of 2109 bp encoding 703 amino acids. The chicken AR (cAR) shares high homology with ARs from other species in its amino acid sequences, in particular DNA binding domain (DBD) and ligand binding domain (LBD). RT-PCR analysis revealed that cAR mRNA is expressed in several embryonic tissues of both sexes, and relatively higher expression was observed in left ovary compared with testis. The immunoreactive signal of AR was co-localized within the ovarian cell nucleus, while such nuclear localization was not detected in those of testis. To get insight on the possible role of androgen-AR signaling during gonadal development, non-steroidal AR antagonist, flutamide, was administrated in ovo. The treatment induced the disorganization of sex cords in ovarian cortex at day 12 of incubation. The effect was restored by testosterone co-treatment, implying the possibility that AR mediated signaling may be involved in ovarian morphogenesis. Furthermore, co-treatment of flutamide with estradiol-17beta (E2) also restored the phenotype, suggesting androgen-AR signaling might activate aromatase expression that is necessary for estrogen synthesis. These findings suggest androgen-AR signaling might contribute to chicken embryonic ovarian development.

Amino Acid Sequence↗

PubMed2000

The human RGL (RalGDS-like) gene: cloning, expression analysis and genomic organization.

Ral GDP dissociation stimulator (RalGDS) and its family members RGL, RLF and RGL2 are involved in Ras and Ral signaling pathways as downstream effector proteins. Here we report the precise localization and cloning of two forms of human RGL gene differing at the amino terminus. Transcript A, cloned from liver cDNA libraries has the same amino terminus as the mouse RGL, whereas transcript B cloned from brain has a substitution of 45 amino acids for the first nine amino acids. At the genomic level, exon 1 of transcript A is replaced by two alternative exons (1B1 and 1B2) in transcript B. Both forms share exons 2 through 18. The human RGL protein shares 94% amino acid identity with the mouse protein. Northern blot analysis shows that human RGL is expressed in a wide variety of tissues with strong expression being seen in the heart, brain, kidney, spleen and testis.

Amino Acid Sequence↗

PubMed2000

Quantitative expression analysis of the small conductance calcium-activated potassium channels, SK1, SK2 and SK3, in human brain.

Small conductance calcium-activated potassium (SK) channels are important in controlling neuronal excitability and three SK channels have been identified to date. In the present study, we report the first quantitative analysis of SK1, SK2 and SK3 expression in human brain using TaqMan RT-PCR on a range of human brain and peripheral tissue samples. SK1 expression is restricted to the brain whereas SK2 and SK3 are more widely expressed.

Brain Chemistry↗