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At least 199 records · Page 11Linked to original sources
PubMed1998

Molecular cloning, functional characterization and mRNA expression analysis of the murine chemokine receptor CCR6 and its specific ligand MIP-3alpha.

We have cloned the murine CCR6 receptor and its ligand, the beta-chemokine mMIP-3alpha. Calcium mobilization assays performed with mCCR6 transfectants showed significant responses upon addition of mMIP-3alpha. Murine MIP-3alpha RNA is expressed in thymus, small intestine and colon, whereas mCCR6 RNA is expressed in spleen and lymph nodes. RT-PCR analysis of FACS-sorted lymphoid and antigen presenting cell subsets showed mCCR6 expression mainly in B cells, CD8- splenic dendritic cells and CD4+ T cells. The cloning and functional characterization of the mCCR6 and mMIP-3alpha will allow the study of the role of these proteins in mouse models of inflammation and immunity.

Amino Acid Sequence↗
PubMed2000

Characterization of the potato MADS-box gene STMADS16 and expression analysis in tobacco transgenic plants.

A new MADS-box gene, STMADS16, has been cloned in Solanum tuberosum L. that is expressed in all vegetative tissues of the plant, mainly in the stem, but not in flower organs. STMADS16 expression is established early during vegetative development and is not regulated by light. Sequence similarity besides the spatial and temporal expression patterns allow to define a novel MADS-box subfamily comprising STMADS16 and the gene STMADS11. Expression of the STMADS16 sense cDNA under the control of the 35S cauliflower mosaic virus promoter modifies the inflorescence structure by increasing both internode length and flower proliferation of the inflorescence meristems, and confers vegetative features to the flower. Moreover, STMADS16 ectopic expression overcomes the increase in flowering time and node number produced under short-day photoperiod, while the flowering time is not affected in long-day conditions. These results are discussed in terms of a possible role for STMADS16 in promoting vegetative development.

Amino Acid Sequence↗
PubMed1999

Mutational and expression analysis of the p73 gene in melanoma cell lines.

A novel p53-related gene, p73, was recently isolated and cytogenetically mapped to chromosome region 1p36. Functionally, p73 expression induces p21waf and suppresses tumor cell growth. We mapped p73 using radiation hybrids and localized the gene to an interval that putatively harbors a melanoma tumor suppressor locus. We then analyzed p73 transcripts from 24 melanoma cell lines using reverse transcription-PCR/single strand conformation polymorphism and identified nine polymorphic sequence changes (three novel and six previously published polymorphisms); furthermore, we found evidence of biallelic transcription in our cell lines. However, we did not detect any deleterious mutations. These data suggest that the p73 gene is unlikely to be essential in melanoma tumorigenesis.

Chromosome Mapping↗
PubMed1996

Expression analysis of an Arabidopsis C2H2 zinc finger protein gene.

C2H2 zinc finger protein genes encode nucleic acid-binding proteins involved in the regulation of gene activity. AtZFP1 (Arabidopsis thaliana zinc finger protein 1) is one member of a small family of C2H2 zinc finger-encoding sequences previously characterized from Arabidopsis. The genomic sequence corresponding to the AtZFP1 cDNA has been determined. Molecular analysis demonstrates that AtZFP1 is a unique, intronless gene which encodes a 1100 nucleotides mRNA highly expressed in roots and stems. A construct in which 2.5 kb of AtZFP1 upstream sequences is linked to the beta-glucuronidase gene was introduced into Arabidopsis by Agrobacterium-mediated transformation of roots. Histochemical analysis of transgenic Arabidopsis carrying the AtZFP1 promoter: beta-glucuronidase fusion shows good correlation with RNA blot hybridization analysis. This transgenic line will be a useful tool for analyzing the regulation of AtZFP1 to further our understanding of its function.

Amino Acid Sequence↗
PubMed1998

Expression analysis of human Rhesus blood group antigens by gene transduction into erythroid and non-erythroid cells.

Rh blood group antigens are associated with non-glycosylated human erythrocyte membrane proteins encoded by two closely related genes, RHCE and RHD, and with a glycoprotein, a critical co-expressing factor encoded by the RH50 gene. The sequence analysis of RHCE transcripts has revealed that RhE/e and C/c serological phenotypes are associated with a nucleotide substitution in exon 5 and six substitutions in exons 1 and 2 of RHCE gene, respectively. Smythe et al. have shown that the full length transcript of RhcE gene expressed c and E antigens and the transcript of RhD gene expressed D and G antigens, using retroviral-mediated gene transduction into K562 cells. We performed an epitope analysis of Rh antigen by constructing retroviral gene coding six RH cDNAs, which contain RhcE, ce, CE and D cDNAs, and CE-D, D-CE chimera cDNAs. The cDNAs were introduced into KU812E cells and the expressed antigens were analyzed by flow cytometry. These studies revealed that the C/c and E/e associated substitutions actually participated in respective polymorphic epitopes. However, the C antigen was not detected on the KU812E cells introduced with CE cDNA, despite E antigen being expressed. The study with the chimera gene between CE and D cDNAs also indicated that the Rh epitopes were not constructed with short polymorphic exofacial peptide loops only but also with other peptide fragments and membrane components. Co-expression studies of Rh50 and RhD or cE gene in non-erythroid cells, 293, and expression studies of Rh50 in another erythroid cell, HEL, did not show any Rh antigens on the transduced cells, despite the Northern blot study showing both transcripts in the cells. It was suggested that at least a second co-expressing factor was needed to express RhCE or D antigens on the plasma membrane.

Cell Line↗
PubMed2000

RNA expression analysis using a 30 base pair resolution Escherichia coli genome array.

We have developed a high-resolution "genome array" for the study of gene expression and regulation in Escherichia coli. This array contains on average one 25-mer oligonucleotide probe per 30 base pairs over the entire genome, with one every 6 bases for the intergenic regions and every 60 bases for the 4,290 open reading frames (ORFs). Twofold concentration differences can be detected at levels as low as 0.2 messenger RNA (mRNA) copies per cell, and differences can be seen over a dynamic range of three orders of magnitude. In rich medium we detected transcripts for 97% and 87% of the ORFs in stationary and log phases, respectively. We found that 1, 529 transcripts were differentially expressed under these conditions. As expected, genes involved in translation were expressed at higher levels in log phase, whereas many genes known to be involved in the starvation response were expressed at higher levels in stationary phase. Many previously unrecognized growth phase-regulated genes were identified, such as a putative receptor (b0836) and a 30S ribosomal protein subunit (S22), both of which are highly upregulated in stationary phase. Transcription of between 3,000 and 4,000 predicted ORFs was observed from the antisense strand, indicating that most of the genome is transcribed at a detectable level. Examples are also presented for high-resolution array analysis of transcript start and stop sites and RNA secondary structure.

Escherichia coli↗
PubMed2000

Cloning, expression analysis, and chromosomal localization of murine and human homologues of a Xenopus mix gene.

We report the cloning and chromosomal localization of murine and human Mix genes, members of a subclass of paired-like homeobox genes of which the Xenopus laevis Mix.1 gene is the founding member. The murine Mix gene was mapped to the distal region of chromosome 1 and the human region to the syntenic region 1q41-42. Northern analysis and RT-PCR of murine adult and embryonic tissues demonstrated that Mix expression was restricted to the early embryo. Whole-mount in situ hybridization revealed patchy but symmetrical Mix expression in visceral endoderm of embryonic day (E)5.5 embryos. In slightly older embryos, the expression was skewed to one side of the embryo and by E6.5, at the onset of gastrulation, expression was seen in the epiblast, visceral endoderm, nascent mesoderm, and the primitive streak. This expression pattern was maintained in mid- and late-streak embryos. In early bud-stage embryos, expression was strongest in the proximal two thirds of the streak, extending to the base of the allantois. By the headfold-stage, expression was confined to the remnant of the primitive streak in the caudal region of the embryo and, after E8.0, in the caudal notochord and tail bud mesoderm. Mix transcripts were no longer detectable after embryonic day 9.5.

Amino Acid Sequence↗
PubMed1998

Cloning and expression analysis of vacuolar H+-ATPase 69-kDa catalytic subunit cDNA in citrus (Citrus unshiu marc.)1.

To investigate the mechanism of sugar accumulation in fruit vacuoles, a full length cDNA (CitVATP-A) encoding the vacuolar H+-ATPase 69-kDa catalytic subunit was isolated from a cDNA library constructed from citrus fruit (Citrus unshiu Marc.). A 2304-bp insert of CitVATP-A was coded for a 623 amino acid polypeptide with a predicted molecular mass of 68.68 kDa. The deduced amino acid sequence for CitVATP-A showed a 96.5% homology with the carrot homologue. Genomic Southern blot analysis suggested that CitVATP-A is a low-copy number gene. Northern blot analysis of leaves and fruits during the developing stages showed that the level of expression is high in young leaves and is low in mature leaves, and that it increased in both the edible parts and the peel, during fruit growth and maturity.

Amino Acid Sequence↗
PubMed1999

Expression analysis of a cytosolic glutamine synthetase gene in cotyledons of Scots pine seedlings: developmental, light regulation and spatial distribution of specific transcripts.

The expression of a cytosolic glutamine synthetase (GS1; EC 6.3.1.2) gene was examined in cotyledons of Scots pine seedlings. Light strongly stimulated GS1 mRNA accumulation during development. Similarly, steady-state levels of GS1 transcripts increased in dark-grown seedlings transferred to light and decreased in dark-adapted seedlings. Light/dark adaptation affected rbcS and lhcb2 mRNA levels and chlorophyll contents in the same manner. Light-grown seedlings in the presence of the herbicide norflurazon showed a drastic decrease in mRNA for GS and photosynthetic proteins, whereas the effect of the herbicide on mitochondrial beta-ATP synthase mRNA was limited. These results indicate that factors associated with developing chloroplasts could be required for maximal GS1 gene expression during seedling development. The level of GS polypeptide, determined by immunoblot, was up-regulated during seedling development in the light or dark. However, the levels of the polypeptide detected were unaltered by the light/dark adaptation treatments. The analysis of GS1 mRNA association with polysomes indicated that the discrepancies between GS protein and mRNA levels are not a result of a differential translational rate of the transcript in darkness relative to light. Two GS isoproteins with different isoelectric point were resolved by two-dimensional PAGE in light- and dark-germinated plants. The relative abundance of one of them was markedly affected by light and correlated with the observed changes in GS mRNA, suggesting that the other form is not a product derived from the detected transcript. In situ hybridization of cotyledon sections showed the presence of GS1 mRNAs in mesophyll and phloem cells confirming gene expression in photosynthetic tissues. High levels of transcript were detected also in meristematic cells of apical primordia. These data suggest a dual role for the GS1 gene associated with chloroplast development/activity and glutamine biosynthesis for nitrogen mobilization during early growth of Scots pine.

Chloroplasts↗
PubMed1997

Isolation of a pancreas-specific gene located on human chromosome 14q31: expression analysis in human pancreatic ductal carcinomas.

We have isolated a novel cDNA (SEL1L) that shows sequence similarities to SEL-1, a gene identified as an extragenic suppressor of the lin-12 hypomorphic mutant from Caenorhabditis elegans (7, 8). SEL1L exhibits a tissue-specific pattern of expression: a single poly(A)+ RNA species of 7.5 kb is abundantly expressed only in the pancreas of healthy individuals, whereas low to undetectable levels are observed in other adult and in some fetal tissues. Somatic hybrid panel and fluorescence in situ hybridization positioned this gene in the q31 band of human chromosome 14. The tissue-specific expression of this gene induced us to study its role in human pancreatic carcinomas. Our analysis revealed that 17% of adenocarcinomas of the pancreas did not express SEL1L to a detectable level; however, no gross genomic alterations were apparent in the few hundred kilobases of the relevant region.

Adult↗
PubMed2000

Expression analysis and mutational screening of the epithelium-specific ets gene-1 (ESE-1) in patients with squamous anal cancer.

To investigate whether ESE-1 gene abnormalities are involved in alterations of epithelial cell differentiation in squamous anal cancer ESE-1 expression and structure were screened in six patients by reverse transcriptase-polymerase chain reaction (RT-PCR) and automated sequence analysis. The complete cDNA of isoform ESE-1b was always expressed and correctly spliced, with single nucleotide polymorphism being observed in two cases. Presence of ESE-1b point mutations was excluded. Expression of SPRR2A and ENDOA/CK8, two epithelium-specific ESE-1 target genes, were revealed by RT-PCR in all cases. This first report of expression of ESE-1, and of SPRR2A and ENDOA/CK8 (both related to terminal differentiation in different types of epithelia lining) in anal cancer excludes the hypothesis that these genes influenced carcinogenesis in our patients. Despite selecting of patients without clinical evidence of HPV infection, PCR consistently revealed HPV-16 DNA, highlighting the importance of HPV infection in anal cancer.

Adult↗
PubMed2000

Gene-expression analysis at the single-cell level.

The manner in which a cell responds to and influences its environment is ultimately determined by the genes that it expresses. To fully understand and manipulate cellular function, identification of these expressed genes is essential. Techniques such as RT-PCR enable examination of gene expression at the tissue level. However, the study of complex heterogeneous tissue, such as the CNS or immune system, requires gene analysis to be performed at much higher resolution. In this article, the various methods that have been developed to enable RT-PCR to be performed at the level of the single cell are reviewed. In addition, how, when carried out in combination with techniques such as patch-clamp recording, single-cell gene-expression studies extend our understanding of biological systems is discussed.

Cell Physiological Phenomena↗
PubMed1999

Expression analysis of the thyrotropin-releasing hormone receptor (TRHR) in the immune system using agonist anti-TRHR monoclonal antibodies.

Monoclonal anti-rat thyrotropin-releasing hormone (TRH) receptor (TRHR)-specific antibodies (mAb) were generated by immunization with synthetic peptides of rat TRHR partial amino acid sequences; one (TRHR01) was directed against a sequence (84-98) in the extracellular portion of the rat TRHR reported to be constant among different species, including man, and the second (TRHR02) recognizes the C-terminal region sequence 399-412. In lysates from GH4C1 cells, a clonal rat pituitary cell line, both mAb recognize the TRHR in Western blot analysis, and TRHR02 immunoprecipitates the TRHR. Incubation of GH4C1 cells with the mAb causes a fluorescence shift in fluorescence-activated cell sorting analysis. The cells were stained specifically by both mAb using immunocytochemical techniques. Furthermore, TRHR01 is agonistic in its ability to trigger Ca2+ flux, and desensitizes the TRH receptor. We tested for TRHR in several rat organs and found expression in lymphoid tissues. TRHR01 recognizes the human TRHR, and analysis of human peripheral blood lymphocyte and tonsil-derived leukocyte populations showed receptor expression in non-activated and phytohemagglutinin-activated T and B cells.

Animals↗
PubMed1998

Probing lymphocyte biology by genomic-scale gene expression analysis.

The identity and abundance of mRNA species within a cell dictate, to a large extent, the biological potential of that cell. Although posttranscriptional mechanisms modify protein expression in critical ways, cellular differentiation requires key changes in gene transcription, as evidenced by the potent phenotypes that result from disruption of transcription factor genes in mice. It is now possible to assess the mRNA profile of a cell globally using recently developed genomics techniques. This review focuses on the potential of cDNA microarrays to define gene expression in lymphoid cells, a field which is in its infancy. Examples of cellular activation genes and cytokine inducible genes discovered using this technology are presented but these represent only a taste of the fruit that this new technology will ultimately bear. Gene expression profiles should provide essential new insights into lymphocyte differentiation and activation, the pathogenesis of immune disorders, and the molecular abnormalities in lymphoid malignancies.

DNA, Complementary↗
PubMed1998

Mutation and expression analysis of the putative prostate tumour-suppressor gene PTEN.

The chromosomal region 10q23-24 is frequently deleted in a number of tumour types, including prostate adenocarcinoma and glioma. A candidate tumour-suppressor gene at 10q23.3, designated PTENor MMAC1, with putative actin-binding and tyrosine phosphatase domains has recently been described. Mutations in PTEN have been identified in cell lines derived from gliomas, melanomas and prostate tumours and from a number of tumour specimens derived from glial, breast, endometrial and kidney tissue. Germline mutations in PTEN appear to be responsible for Cowden disease. We identified five PTEN mutations in 37 primary prostatic tumours analysed and found that 70% of tumours showed loss or alteration of at least one PTEN allele, supporting the evidence for PTEN involvement in prostate tumour progression. We raised antisera to a peptide from PTEN and showed that reactivity occurs in numerous small cytoplasmic organelles and that the protein is commonly expressed in a variety of cell types. Northern blot analysis revealed multiple RNA species; some arise as a result of alternative polyadenylation sites, but others may be due to alternative splicing.

Amino Acid Sequence↗
PubMed1998

Characterisation of the coding sequence and fine mapping of the human DFFRY gene and comparative expression analysis and mapping to the Sxrb interval of the mouse Y chromosome of the Dffry gene.

DFFRY (the Y-linked homologue of the DFFRX Drosophila fat-facets related X gene) maps to proximal Yq11.2 within the interval defining the AZFa spermatogenic phenotype. The complete coding region of DFFRY has been sequenced and shows 89% identity to the X-linked gene at the nucleotide level. In common with DFFRX , the potential amino acid sequence contains the conserved Cys and His domains characteristic of ubiquitin C-terminal hydrolases. The human DFFRY mRNA is expressed in a wide range of adult and embryonic tissues, including testis, whereas the homologous mouse Dffry gene is expressed specifically in the testis. Analysis of three azoospermic male patients has shown that DFFRY is deleted from the Y chromosome in these individuals. Two patients have a testicular phenotype which resembles Sertoli cell-only syndrome, and the third diminished spermatogenesis. In all three patients, the deletions extend from close to the 3' end into the gene, removing the entire coding sequence of DFFRY. The mouse Dffry gene maps to the Sxrb deletion interval on the short arm of the mouse Y chromosome and its expression in mouse testis can first be detected between 7.5 and 10.5 days after birth when type A and B spermatogonia and pre-leptotene and leptotene spermatocytes are present.

Adult↗
PubMed1997

Cloning and expression analysis of mouse Cclp1, a new gene encoding a coiled-coil-like protein.

Here we describe the nucleotide sequence and expression pattern of a novel gene termed Coiled-coil-like protein 1 (Cclp1). A 2646bp open reading frame encodes a 882 amino acid protein with a predicted coiled-coil domain at the amino terminus. Cclp1 is expressed in a variety of adult tissues and during different stages of embryogenesis. The broad expression pattern suggests a general cellular function of CCLP1.

Amino Acid Sequence↗
PubMed2026

Zea mays Drought-Overly Sensitive1/TUBA4 Is Wilty3, and Transcriptome Co-Expression Analysis of Shoot Meristem Mutant Tissues Reveals Wilty2/TUB6:Wi3 Interactions Associated With Stem Vascular Bundle Development.

Plant vasculature is essential for the transport of water, nutrients, and signaling molecules across organs, while also providing critical mechanical support for growth and development. Disruptions in vascular bundle formation can therefore lead to severe physiological and developmental defects. In maize, ethyl methanesulfonate (EMS)-induced dominant nonallelic Wilty mutants exhibit a pronounced wilting phenotype even under well-watered conditions, indicating underlying defects in vascular function. In this study, we characterized the Wi3 mutant, identified as ZmDrought-Overly-Sensitive1/DOS1, and compared it with the previously described Wi2 mutant to uncover shared mechanisms underlying their phenotypes. We provide evidence, by bulk segregant resequencing linkage disequilibrium of SNPs adjacent to the causal Wilty SNPs in respective ß- and α-tubulin genes, for the personal communication from Gerry Neuffer that Wi2/ß-tub6 provenance is from ACR-related stock, whereas Wi3/α-tub4 allele is from Mo17, not B73 as claimed by the authors who cloned Dos1. Histochemical staining and Fourier-transform infrared (FTIR) spectroscopy of vascular bundles in Wi3 indicated apparent alterations in cellulose and lignin content consistent with those observed in Wi2. Transcriptome analysis of shoot meristems further indicated that similar sets of genes and pathways are differentially expressed in both mutants, suggesting convergence on common biological pathways. Using bulk-segregant whole-genome resequencing, we identified alpha-tubulin4 (TUA4) as the causal gene in Wi3 (ZmDOS1), harboring a C-to-T substitution within the N-terminal GTPase-binding domain. This mutation results in a glutamic acid196-to-lysine substitution. Given that α- and β-tubulin subunits heterodimerize, and in many plants and animal mutant alleles are dominant-negative gains-of-function, we infer Wi2, Wi3, and likely Wi4, based on very similar FTIR biophysical difference spectra, may act as effectors of vascular bundle cell wall deposition, potentially involving vesicle trafficking as recently shown for asymmetric cell divisions in maize stomatal development. Together, these findings highlight the functional interdependence of tubulin subunits and provide a plausible mechanistic framework for the striking biophysical, transcriptomic, and phenotypic similarities observed between Wi2, Wi3/ZmDOS1, and Wi4 mutants.

bulk segregant analysis↗