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PubMed1989

Regulation of IL-1 and TNF-alpha expression during the differentiation of bone marrow derived macrophage.

Macrophage differentiation is accompanied by the acquisition of both Ag presentation and tumoricidal activities. In this set of experiments, the expression of IL-1 by bone marrow-derived macrophage (BMDM) was found to be highly regulated, with both the expression of IL-1 mRNA and mIL-1 appearing only at discrete stages of activation. The accumulation of IL-1 alpha (membrane) mRNA was induced by endotoxin but not IFN-gamma or CSF-1. mIL-1 was detected by D10.G4.1 T cells on BMDM only after 7 days of in vitro differentiation. Secreted IL-1 beta was detected by day 3 of culture, with enhanced production observed after activation with endotoxin. IL-1 beta mRNA was found to be constitutively expressed in BMDM as early as day 3 of culture. The expression of IL-1 beta mRNA was up-regulated by endotoxin after 30 min of exposure with maximal expression occurring after 2 to 6 h of exposure. Constitutive expression of IL-1 alpha mRNA was not detected but 1 h of endotoxin exposure resulted in the appearance of IL-1 alpha transcripts. As with IL-1 beta, TNF-alpha mRNA was also constitutively expressed during a wide time period of differentiation; however, in contrast, to IL-1 beta, TNF-alpha mRNA expression was up-regulated by both endotoxin and IFN-gamma. The expression of TNF-alpha macrophage by BMDM coincided with the acquisition of tumoricidal activity. An examination of the mRNA sequences encoding the proto-oncogenes c-myc and c-fms demonstrated the expression of c-myc only on day 3, whereas c-fms was constitutively expressed throughout the culture period. Endotoxin stimulation of BMDM resulted in a transitory increase in c-myc expression only at day 3 of culture, whereas endotoxin had no effect on c-fms expression until 7 days of culture at which time expression declined. In contrast, the expression of transferrin receptor mRNA transcripts, which were also constitutively expressed throughout the entire culture period, were not affected by stimulation with either endotoxin or IFN-gamma. These results indicate BMDM expression of the affector molecules IL-1 alpha and beta and the effector molecule TNF-alpha are regulated separately during unique "differentiation-specific" phases of development.

Animals↗
PubMed2004

Cloning and expression analysis of cDNAs corresponding to genes activated in cucumber showing systemic acquired resistance after BTH treatment.

BACKGROUND: Infection of plants by necrotizing pathogens can lead to the rapid and localized induction of a complex set of defense responses resulting in a restriction of pathogen growth and spread. Subsequently, an increase of plant resistance against a broad spectrum of pathogens is observed systemically. This plant immunity is known as Systemic Acquired Resistance. To identify components of the transduction pathway, we cloned and analysed the expression pattern of several mRNAs accumulating in cucumber plants after induction of Systemic Acquired Resistance. RESULTS: We tested on cucumber different compounds known to induce systemic acquired resistance. Among these, BTH (benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester) proved to be very effective. mRNA RT-PCR differential display was used to identify mRNA sequences induced 24 hours after the application of 10 microM BTH to cucumber plants. A cDNA library constructed from cucumber plants sprayed with 10 microM BTH was screened to get corresponding full length cDNAs. Among the identified cDNAs were those coding for a putative ras-related GTP-binding protein, a putative beta-1,4-N-Acetylglucosaminyltranferase III and a putative pathogenesis related protein. The time course of accumulation of the three corresponding mRNAs was analysed by northern blotting in plants treated by BTH or in plants infected by Colletotrichum lagenarium. CONCLUSIONS: The mRNA RT-PCR differential display technique allowed the identification of three genes possibly involved in Systemic Acquired Resistance in cucumber. Pathogenesis-related proteins are known to be involved in plant defence against pathogens. GTP-binding protein and N-acetylglucosaminyltranferase III have been reported to be components of signal transduction pathways in mammals and plants.

Blotting, Northern↗
PubMed1988

The human protein S locus: identification of the PS alpha gene as a site of liver protein S messenger RNA synthesis.

The protein S locus, situated on chromosome 3, consists of two protein S genes. Here, we report the cloning and complete nucleotide sequence of the 3'-untranslated region of the two genes designated PS alpha and PS beta. Both regions span approximately 1,200 nucleotides. They show a high degree (-97%) of homology, with deviations caused by small deletions, insertions and point mutations. Comparison of PS alpha and PS beta with the reported protein S liver cDNAs, shows that the latter all originate from the PS alpha gene. The PS alpha gene therefore is marked as the major site of synthesis of liver protein S mRNA. Sequence comparison with the bovine protein S cDNA reveals that the PS beta gene has accumulated a few more mutations than the PS alpha gene since duplication of the ancestral protein S gene that seems to have occurred recently during primate evolution.

Animals↗
PubMedDate not supplied

A family of cloning vectors containing the lacUV5 promoter.

A family of plasmids containing short pieces of Escherichia coli lac promoter DNA has been constructed. DNA fragments from any source may be inserted directly into the unique EcoRI sites of some of these plasmids to achieve transcription under the control of the lacUV5 promoter. Alternatively, the plasmids serve as convenient sources of lac DNA fragments ('portable promoters') containing the 'up' promoter mutations UV5 or Ps (super promoter) as well as the wild-type promoter. pOP95-2, pOP95-5, pOP203-1, pOP203-2 and pOP203-3 are derivatives of pMB9 while pOP95-15 and pOP203-13 are derivatives of pBR322. The pOP95 plasmids contain the 95-bp AluI lac fragment. This fragment includes the UV5 promoter (minus the CAP binding site), the repressor binding site, and ends 2 bp before an ATG encoding the beta-Gal start codon. The pOP203 plasmids contain the 203-bp HaeIII lac fragment. This fragment contains the UV5 promoter (including the L8 mutation in the CAP binding site), the repressor binding site and sequences encoding the first 8 amino acids of beta-Gal. To shorten and introduce reading frame heterogeneity in the beta-Gal coding end of the pOP203 plasmids, the EcoRI site in pOP203-12 was moved upstream by digesting EcoRI cut plasmid DNA with T4 DNA polymerase and S1 nuclease followed by ligation in the presence of EcoRI linker. This produced the plasmids pOP203-24, pOP203-27, pOP203-28 and pOP203-29. pOP203-29 encodes essentially just that portion of the beta-Gal mRNA sequence which is protected from nuclease digestion by the bound ribosomal complex (Maizels, 1974).

Amino Acid Sequence↗
PubMed1999

Hypoxia alters iron-regulatory protein-1 binding capacity and modulates cellular iron homeostasis in human hepatoma and erythroleukemia cells.

Ferritin and transferrin receptor expression is post-transcriptionally regulated by a conserved mRNA sequence termed the iron-responsive element (IRE), to which a transacting protein called the iron-regulatory protein (IRP) is bound. Our data demonstrate that hypoxia powerfully enhances IRE/IRP-1 binding in human cell lines. Using the human hepatoma cell line Hep3B as a model, we found that 16 h in a 1% oxygen atmosphere markedly increases IRE/IRP-1 binding as assessed by electromobility shift assay. Hypoxia also decreased cytosolic aconitase activity. The hypoxia-enhanced IRE/IRP-1 binding stabilized the transferrin receptor message, increased the cellular mRNA content by over 10-fold, and doubled surface receptor expression. Simultaneously, hypoxia suppressed ferritin message translation. Hypoxia's effect was most strikingly depicted by the absence of ferritin synthesis in cells challenged with inorganic iron. Our results contrast with previously reported data (Hanson, E. S., and Leibold, E. A. (1998) J. Biol. Chem. 273, 7588-7593) in which a 3% oxygen atmosphere reduced IRE/IRP-1 binding in rat hepatoma cells. We discuss some possible reasons for the differences. In aggregate with other investigations involving responses to hypoxia, iron, or nitric oxide, our data indicate that cellular iron metabolic responses are complex and that IRE/IRP-1 interactions vary between cell lines and perhaps between species.

Animals↗
PubMed2000

Bovine NAD+-dependent isocitrate dehydrogenase: alternative splicing and tissue-dependent expression of subunit 1.

NAD+-dependent isocitrate dehydrogenase (IDH), a key regulatory enzyme in the Krebs cycle, is a multi-tetrameric enzyme. At least three of the subunits in the core tetramer of mammals are unique gene products. Subunits 1/beta and 2/gamma are considered to be regulatory, while subunits 3,4/alpha, comprising half the tetramer, are catalytic. The full sequence was obtained for the major subunit 1 cDNA in bovine heart, IDH 1-A. A second cDNA, rare in heart, was also identified (IDH 1-B). Differences in the two were confined to the 3'-region, suggesting alternative splicing. Screening of brain, kidney, and liver RNA showed the presence of IDH 1-A and 1-B and a third major species, IDH 1-C. Amplification of bovine genomic DNA by PCR across the regions of difference produced a single product. Comparison of the genomic and mRNA sequences showed that IDH 1-A resulted from splicing of exon W to exon Y, eliminating intron w, exon X, and intron x. IDH 1-B was formed by splice junctions between exon W, exon X, and exon Y. IDH 1-C resulted from splicing of exon W to exon X and subsequent retention of intron x. The 2 proteins predicted from these 3 mRNAs are identical over their first 357 residues. Protein IDH 1-A, resulting from a termination codon within exon Y, contains an additional 26 residues. Proteins IDH 1-B and 1-C derive from a common termination codon within exon X and contain an additional 28 residues. The two C-terminal regions differ notably in the number and nature of charged residues, resulting in proteins with a charge difference of 3.2 at pH 7.0. Subunit 1 sequences previously reported from other species grouped with one or the other of the bovine proteins. No evidence was found for alternative splicing in subunit 3,4/alpha. The results of the present study, together with recent work on the 2/gamma subunit [Brenner,V., Nyakatura, G., Rosenthal, A., and Platzer, M. (1998) Genomics 44, 8], indicate that the regulatory subunits of the enzyme, but not the catalytic, possess alternatively spliced forms varying in C-terminal properties with tissue-specific expression. The finding is suggestive of a mechanism for modulation of allosteric regulation tailored to the needs of different tissues.

Alternative Splicing↗
PubMed1997

Antitermination of transcription of catabolic operons.

Antitermination of transcription mediated by proteins interacting with mRNA sequences is described for nine operons/regulons. Eight of the systems are catabolic, while the ninth, the Klebsiella pneumoniae nas regulon, is involved in the assimilation of nitrate and nitrite. Six of the catabolic operons/regulons are found in Bacillus subtilis, one is found in Escherichia coli, and one in Pseudomonas aeruginosa. The antitermination system of five of the operons/regulons (E. coli blg, and sacPA, sacB, bgl, and lic from B. subtilis) are assigned to the bgl-sac family on the basis of extensive similarities with regard to antiterminator proteins and the sequences of the antiterminators. Other members of the bgl-sac family are the arb operon of Erwinia chrysanthemi and a presumed bgl operon of Lactococcus lactis. The antitermination systems of the other four operons/regulons (B. subtilis glp, B. subtilis hut, P. aeruginosa ami, and K. pneumoniae nas) seem to be unrelated both to the bgl-sac family and to each other. The antiterminator protein of the B. subtilis glp regulon has been found not only to cause antitermination but also to stabilize the resultant mRNA and to mediate glucose repression. If other antiterminator proteins, and antitermination factors, also prove to have additional functions, it will broaden the impact of antitermination as a means of controlling gene expression.

Bacillus subtilis↗
PubMed1993

Molecular homology among members of the R gene family in maize.

The R gene family determines the timing, distribution and amount of anthocyanin pigmentation in maize. This family comprises a set of regulatory genes, consisting of a cluster of several elements at the R locus, on chromosome 10, the Lc and Sn gene lying about two units R distal and B on chromosome 2. Each gene determines a tissue-specific pigmentation of different parts of the seed and plant. The proposed duplicated function of R, Sn, Lc and B loci is reflected in cDNA sequence similarity. In this paper an extensive analysis of the predicted proteins of the R, Sn, Lc and B genes together with a search for putative sites of post-translational modification is reported. A comparison with the prosite database discloses several N-glycosylation and phosphorylation sites, as well as the basic Helix-Loop-Helix (HLH) domain of transcriptional activators. Sn, Lc, and R-S show a high conservation of these sites, while B is more divergent. Analysis of the 5' leader of mRNA sequences discloses the presence of five ATG triplets with two upstream open reading frames (uORFs) of 38 and 15 amino acids and a loop structure indicating a possible mechanism of control at the translational level. It is conceivable that possible mechanisms acting at the translational and post-translational level could modulate the expression and the activation of these transcription factors. Northern analysis of various tissues of different R alleles highlights a strict correlation between pigment accumulation in different tissues and the expression of the regulatory and structural genes suggesting that the pattern of pigmentation relies on a mechanism of differential expression of the members of the R family. Analysis of the Sn promoter discloses the presence of several sequences resembling binding sites of known transcription factors (as GAGA and GT) that might be responsible for the spatial and light-induced expression of this gene. Two regions include a short sequence homologous to the consensus binding site of the B-HLH domain suggesting a self-regulatory control of the Sn gene.

Amino Acid Sequence↗
PubMed1984

Site specific deletions of regulatory sequences in a ribosomal protein-RNA polymerase operon in Escherichia coli. Effects on beta and beta' gene expression.

The 319 nucleotide long intergenic region between the rplL (L12) and the rpoB (beta) genes of the L10 operon contains a transcription attenuation sequence and a RNase III mRNA processing sequence. Four site specific deletions located within this intergenic space which remove either the transcription attenuation sequence or the RNase III mRNA processing sequence or both sequences have been isolated on recombinant DNA plasmids carrying this operon. Deletions of sequences surrounding the RNase III processing site result in a uniform 80-90% reduction in the translational efficiency of beta subunit mRNA. This reduction in translation efficiency appears not to be related to processing per se; transcription of the rpoB and rpoC genes and the translation efficiency of the respective mRNA sequences were indistinguishable in an RNase III processing defective mutant (rnc) and its isogenic parent (rnc+). Deletions of the attenuator sequence result in a substantial increase in the downstream transcription of the beta subunit gene. The translational efficiency of RNase III processed beta subunit mRNA was found to be related in an inverse manner to the level of beta subunit synthesis. These result suggest that sequences on the mRNA in the vicinity of the RNase III processing site (i) are essential for efficient translation of beta subunit mRNA and (ii) are utilized for reducing the translational efficiency of the beta subunit mRNA when the beta subunit protein is produced in excess of that required for RNA polymerase assembly.

Base Sequence↗
PubMed2006

Validity of messenger RNA expression analyses of human saliva.

PURPOSE: The origins of expression microarray and reverse transcription-PCR (RT-PCR) signals in human saliva were evaluated. EXPERIMENTAL DESIGN: The "RNA" extracts from human saliva samples were treated with vehicle, DNase, or RNase. Two-step amplification and hybridization to Affymetrix 133A cDNA microarrays were then done. Confirmatory RT-PCR experiments used conventionally designed PCR primer pairs for the reference housekeeper transcripts encoding 36B4, beta-actin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA sequences, which are known to be homologous to genomic DNA pseudogene sequences. Negative controls included the omission of reverse transcriptase ("no-RT") to detect any DNA-derived signal. Finally, an RNA-specific RT-PCR strategy eliminated confounding signals from contaminating genomic DNA. RESULTS: Microarray experiments revealed that untreated, DNase-treated, and RNase-treated "RNA" extracts from saliva all yielded negligible overall signals. Specific microarray signals for 36B4, beta-actin, and GAPDH were low, and were unaffected by RNase. Real-time quantitative RT-PCR reactions using conventional, non-RNA-specific primers on saliva samples yielded PCR products for 36B4, beta-actin, and GAPDH; DNase-treated saliva samples did not yield a PCR product, and the "no-RT" and "+RT" conditions yielded similar amounts of PCR product. The RNA-specific RT-PCR strategy, across all conditions, yielded no PCR product from saliva. CONCLUSIONS: The combination of (a) a minimal microarray signal, which was unaffected by RNase treatment, (b) the presence of a conventional RT-PCR housekeeper product in both RNase-treated and no-RT saliva samples, (c) the absence of a conventional RT-PCR housekeeper product in DNase-treated conditions, and (d) the absence of a RNA-specific RT-PCR product shows that any microarray or RT-PCR signal in the saliva must arise from genomic DNA, not RNA. Thus, saliva extracts do not support mRNA expression studies.

Actins↗
PubMed2005

Application of hammerhead ribozymes in filamentous fungi.

Metabolic engineering in filamentous fungi is a emerging field of research as many fungi produce high value primary and secondary metabolites. Ribozyme technology can be used as a tool for metabolic engineering to influence metabolic pathways and to knock down the expression of specific genes of interest. Hammerhead ribozymes can target virtually any mRNA sequence of choice and prevent gene expression on the post-transcriptional level. They are thus a versatile tool for timed and spatial elimination of unwanted gene products. As current research has only investigated the application of ribozymes in bacteria, yeast and mammalian cells, we decided to carry out a study on whether this technology can also function with filamentous fungi. We employed a sensitive, quantitative reporter-based model system as a proof of concept, using the Escherichia coli beta-glucuronidase transcript (uidA) as the target mRNA and Aspergillus giganteus as the host. This system was used to validate the in vivo activities of seven different hammerhead ribozymes, which were selected by in silico analysis of the uidA mRNA. All ribozymes tested were able to reduce the reporter activity up to a maximum of 100%, demonstrating that ribozyme technology is indeed a useful tool in fungal metabolic engineering.

Aspergillus↗
PubMed2002

A database on alternative splice forms on the integrated genetic map service (IGMS).

UNLABELLED: The IGMS is a comprehensive information system that combines the knowledge from genomic sequence, genetic map and genetic disorders databases. This system is updated weekly and focuses on the analysis of EST data. The IGMS identifies UniGene clusters that are differentially expressed in different types of cancer with respect to different reference tissues. The results can be combined with clinical data to asses the potential relevance of specific genes for patient survival or metastatic spread. The second application maps EST with a specific expression profile. Our third application generates a database of alternative splice forms for nine organisms from EST and mRNA sequence data. The results can be used to find splicing patterns specific for certain tissues or tumour types. AVAILABILITY: http://www.bioinf.mdc-berlin.de/igms/.

Alternative Splicing↗
PubMed1991

In situ hybridization with non-radioactive digoxigenin-11-UTP-labeled cRNA probes: localization of developmentally regulated mouse tenascin mRNAs.

An improved method for in situ hybridization was developed in order to identify the tissue-specific expression of messenger RNA (mRNA) for the novel extracellular matrix glycoprotein, tenascin, during mouse development. Non-radioactive RNA probes were generated by incorporating digoxigenin-11-UTP instead of conventional isotopic labels. Hybridization of anti-sense probes to complementary mRNAs was detected by a chromogenic staining reaction catalyzed by an anti-digoxigenin antibody-alkaline phosphatase conjugate. Markedly improved enhancement of staining was achieved by expanding the complexity of probes and strictly controlling the degree of proteolytic digestion of paraformaldehyde-fixed tissue sections. Six different complementary RNA (cRNA) probes representing most of the tenascin mRNA sequence were prepared. Very weak signals were obtained after single applications of each probe, but strong specific signals were present when all six probes were mixed together. In either case, no signal was found without prior proteolytic digestion of tissue sections with proteinase K. Treatment with increasing concentrations of proteinase K initially resulted in increased sensitivity of signal detection, but extensive digestion resulted in histological sections of poor quality for light microscopy. Optimal conditions varied according to the tissue type examined. In lung, in situ hybridization detected tenascin mRNA in the relatively large cells lining alveolar walls adjacent to type I pneumocytes. In cerebellum, glial cells of the Purkinje cell layer contained tenascin mRNA, but Purkinje cells did not. In both cases, hybridization signals were confined to the cytoplasm of cells, and no extracellular staining was observed. This method provides a promising new tool for analysis of spatio-temporal regulation of tenascin gene expression during embryogenesis and oncogenesis.

Animals↗
PubMed1987

Point mutations as an optimal search process in biological evolution.

Point mutations are pictured as jumps in a phase space representing the sequences of amino acids or nucleotides as discrete points. It is shown that this space can be given a natural metric by quantifying common physical and chemical properties of amino acid constituents in terms of a natural measure. Evolution through point mutations is simulated by the search for points in the phase space representing amino acid sequences of high survival fitness. Due to the local compactness of the distribution of these functionally allowed points in phase space any successful search procedure has characteristics qualitatively different from those in the case of a random distribution. This is demonstrated by model calculations. A specified distribution of allowed points is generated with subsequent evaluation of the success of the retrieval process as a function of the jump probabilities between lattice sites. The results of such simulations are compared with data obtained from the analysis of the DNA or mRNA sequences coding related proteins. By counting silent and expressed nucleotide replacement frequencies one can draw conclusions as to the efficacy of the natural evolutionary search processes in the phase space of amino acid sequences. There are cases, where the highest possible information gain of one bit per accepted point mutation is achieved. In general the information gain is found to be somewhat sub-maximal due to functional requirements.

Amino Acid Sequence↗
PubMed1988

Structural characterization of Dictyostelium discoideum prespore-specific gene D19 and of its product, cell surface glycoprotein PsA.

The Dictyostelium discoideum cell surface antigen PsA is a glycoprotein which first appears in the multicellular stage soon after tip formation and is selectively expressed on prespore cells. The D19 gene encodes an mRNA sequence which is highly enriched in prespore over prestalk cells in the slug stage. We have determined 81 amino acid residues of N-terminal sequence from immunoaffinity-purified PsA protein and shown this sequence to be identical to the predicted sequence of the D19 gene. There are several short repeat elements close to the C terminus, and unequal crossing-over within these is proposed to account for the size polymorphism observed in PsA protein isolated from different D. discoideum strains. The repeats are proline rich and show similarity to the C-terminal region of the D. discoideum cell adhesion molecule, contact sites A. The extreme C terminus, which is also homologous to contact sites A, is characteristic of proteins attached to the plasma membrane via a glycosyl-phosphatidylinositol link. We have marked the PsA gene by insertion of an oligonucleotide encoding an epitope of the human c-myc protein. A construct containing this gene and 990 base pairs of 5'-flanking region directed correct temporal and spatial mRNA accumulation. We found the marked PsA protein, detected with the human c-myc antibody, to be correctly localized on the surface of cells.

Amino Acid Sequence↗
PubMed2000

MEN1 gene mutation analysis of high-grade neuroendocrine lung carcinoma.

Neuroendocrine tumors of the lung consist of a spectrum of neoplasms, including typical carcinoids, atypical carcinoids, large-cell neuroendocrine carcinomas (LCNEC), and small-cell lung carcinomas (SCLC). We previously reported frequent inactivation of the gene responsible for multiple endocrine neoplasia type 1 (MEN1) in both typical and atypical carcinoid tumors. In the present study, we extend the analysis of human NE lung tumors to include 9 primary SCLCs, 36 SCLC cell lines, and 13 primary LCNECs for MEN1 gene inactivation. In SCLC, loss of heterozygosity (LOH) at the MEN1 gene on chromosome band 11q13 was detected in one primary tumor and two cell lines. The coding sequence and splice junctions of the MEN1 gene were screened for mutations in all 44 tumors and cell lines, and no mutations were detected. Northern blot analysis of 13 SCLC cell lines showed the MEN1 transcript to be present and of normal size. In LCNECs, a somatic frameshift in the MEN1 gene (1226delC) was found in one of 13 tumors, representing the first mutation observed outside the spectrum of neoplasms associated with MEN1. Interestingly, neither a deletion nor a mutation was detected in the other allele, and wild-type mRNA sequence was expressed in the tumor, suggesting that the MEN1 gene was not inactivated by a conventional two-hit mechanism. The data support the hypothesis that SCLC and lung carcinoids develop via distinct molecular pathways; however, further investigation is necessary to determine the significance of the MEN1 gene mutation observed in a single case of LCNEC. Published 2000 Wiley-Liss, Inc.

Carcinoma, Large Cell↗
PubMed2004

Application of RNA interference to chicken embryos using small interfering RNA.

Gene silencing using small interfering RNA (siRNA) is not established in avian species. The present study was performed to evaluate RNA interference (RNAi) in the chicken embryo by using a dual fluorescence reporter assay, a plasmid encoding green fluorescent protein (GFP) and a plasmid encoding red fluorescent protein (RFP). The siRNA targeting the GFP mRNA sequence (GFP-siRNA) with both plasmids was introduced into cultured cells and whole embryos by lipofection and microelectroporation, respectively. GFP- and RFP-expressed cells and embryos were observed under fluorescent microscopy and analyzed by flow cytometer, and their mRNAs were analyzed by reverse transcription-PCR (RT-PCR). The strong fluorescence was observed by introducing both plasmids into cells. The intensity of the green fluorescence generated by GFP was greatly suppressed by introducing GFP-siRNA. RT-PCR analysis showed that introducing GFP-siRNA also decreased GFP mRNA levels. In contrast to GFP, the intensity of the red fluorescence generated by RFP and the RFP mRNA levels remained unchanged. In whole embryos, also, introducing GFP-siRNA specifically suppressed GFP expression, and the suppression was maintained for at least 72 h. Consequently, it was concluded that the gene silencing using siRNA is applicable to analyzing the function of genes of interest during avian embryogenesis.

Animals↗
PubMed2002

Beta-catenin mutations correlate with over expression of C-myc and cyclin D1 Genes in bladder cancer.

PURPOSE: We hypothesized that over expression of c-myc and cyclin D1 genes is transcriptionally activated by beta-catenin mutation independent of gene amplification in bladder cancer. To test this hypothesis we investigated the relationship of beta-catenin mutation to c-myc and cyclin D1 mRNA with special reference to the changes in copy number of the 2 genes. MATERIALS AND METHODS: Genomic DNA and total RNA were extracted from 59 bladder cancer specimens and from 31 histologically normal specimens of bladder mucosa. We performed beta-catenin deletion screening by polymerase chain reaction (PCR) using primers spanning exons 3 (including the glycogen synthase kinase-3beta consensus motif), 5 and 6. Mutational changes in beta-catenin in exons 3, 5 and 6 were detected by each PCR-single strand conformational polymorphism analysis followed by direct DNA sequencing. mRNA expression and copy numbers of c-myc and cyclin D1 were determined by semiquantitative reverse transcriptase-PCR and competitive genomic PCR. RESULTS: Missense mutations of beta-catenin found in grade 3 bladder cancer were involved in the consensus motif of glycogen synthase kinase-3beta in exon 3. These cancers showed strong intracellular accumulation of beta-catenin and intense expression of c-myc and cyclin D1 mRNA compared with samples lacking the beta-catenin mutation. When grade 3 cancers were compared, expression levels of c-myc and cyclin D1 mRNA were still higher in those with versus without the beta-catenin mutation. In bladder cancers with beta-catenin mutations copy numbers of the c-myc and cyclin D1 genes did not amplify. CONCLUSIONS: Bladder cancer harboring a beta-catenin mutation may represent aggressive biological behavior with enhanced proliferating activity. These findings are important for understanding the role of beta-catenin mutation in the pathogenesis of bladder cancer.

Carcinoma, Transitional Cell↗