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PubMed1994

Polioviruses containing picornavirus type 1 and/or type 2 internal ribosomal entry site elements: genetic hybrids and the expression of a foreign gene.

A picornavirus hybrid genome was constructed in which the internal ribosomal entry site (IRES) of encephalomyocarditis virus was inserted between the 5' non-translated region and the open reading frame of poliovirus (PV), type 1 (Mahoney). Upon transfection into HeLa cells, the hybrid RNA replicated and yielded a derivative of PV (W1-PNENPO). The PV IRES could be removed from pPNENPO, which resulted in a hybrid picornavirus (W1-P108ENPO) in which the translation of the PV open reading frame normally promoted by the type 1 IRES of PV was promoted by the type 2 IRES of encephalomyocarditis virus. This result indicates that these elements are not likely to contain cis-acting elements necessary for PV replication or encapsidation. A foreign gene (bacterial chloramphenicol acetyltransferase, CAT) was inserted into pPNENPO cDNA between the PV and encephalomyocarditis virus IRES elements. The dicistronic RNA replicated in HeLa cells and yielded a derivative of PV (W1-DICAT) with a genome 17% longer than that of wild-type PV. CAT assays and immunoblot analyses showed that the viral RNA efficiently expressed the foreign gene in cell culture. The CAT activity diminished somewhat with each passage of the dicistronic virus, an observation which suggested that the inserted gene had a deleterious effect on viral replication. However, even after five virus passages, a significant quantity of the foreign gene was still expressed. Insertion of the open reading frame of luciferase (67 kDa) resulted in an RNA species that replicated and expressed luciferase for up to 20 hr after transfection. However, this elongated RNA was not encapsidated.

Base Sequence↗
PubMed1988

Transcriptional activity of human papillomavirus type 6 in respiratory tract papillomata.

We have investigated the molecular basis for differences that we observed in the biological activities of genetically related but distinguishable human papillomavirus type 6 (HPV-6) subtypes. To analyse tissue-specific differences in replication and transcription, and to identify viral gene products important in the benign transformation of epithelial cells, we have modified procedures utilizing guanidinium isothiocyanate and density gradient centrifugation to facilitate the extraction of relatively undegraded DNA and RNA from 21 biopsy specimens of respiratory tract papillomata. Southern transfer analysis was used to characterize the viral genome, and to demonstrate that relative quantities of viral DNA in lesions varied but that the range was similar in lesions induced by HPV-6c, -6d, -6e and -6f. Dot blot analysis of the amount of viral RNA in comparison with the amount of 28S ribosomal RNA indicated that the relative level of viral RNA in each lesion varied considerably and that on average there was approximately twice as much viral RNA in HPV-6c-induced lesions as in HPV-6d-, -6e- or -6f-induced lesions. In dot blot and Northern analyses, hybridization of RNA from HPV-6c-induced lesions with HPV-6c DNA gave a stronger signal than hybridization of the same RNA with an HPV-6e probe, and vice versa. These differences in hybridization intensities with subtype-specific probes indicate that the most abundant RNA species are transcribed from parts of the genome that show sequence divergence between these two subtypes. Northern analysis demonstrated the predominant viral transcript to be about 1200 nucleotides in length in lesions induced by each of the four subtypes.

DNA, Neoplasm↗
PubMedDate not supplied

[Effect of the intake of imbalanced protein on the liver of growing rats].

The goal of the present work was to reproduce in laboratory animals situations frequently observed in low-income population, such as the insufficient energetic ingestion associated with protein sources of inferior quality. Corn was chosen as the protein source of low biological value and casein as the reference protein. The latter was administered in adequate quantity (20%) or equal to that of the corn (10%). Several biochemical parameters and the amounts of DNA, RNA and protein in the liver of rats in a phase of rapid growth were studied. In result, we can attest that the group fed on corn were the most seriously compromised in body weight, liver weight and biochemical parameters with the group fed a restricted diet of corn being the most seriously affected. The results suggest that the alterations caused by ingestion of a balanced diet restricted in quantity were less accentuated that those observed when the diet was of corn "ad libitum".

Animals↗
PubMed1998

Association of CD4 cell depletion and elevated blood and seminal plasma human immunodeficiency virus type 1 (HIV-1) RNA concentrations with genital ulcer disease in HIV-1-infected men in Malawi.

CD4 cell counts and blood plasma and seminal plasma human immunodeficiency virus type 1 (HIV-1) concentrations were compared in HIV-1 RNA-seropositive men with urethritis and with or without genital ulcer disease (GUD). GUD was associated with lower CD4 cell counts (median, 258 vs. 348/microL) and increased blood plasma HIV-1 RNA (median, 240 x 10[3] vs. 79.4 x 10[3] copies/mL). Men with nongonococcal urethritis and GUD shed significantly greater quantities of HIV-1 in semen (median, 195 x 10[3] vs. 4.0 x 10[3] copies/mL) than men with nongonococcal urethritis without GUD. These levels decreased approximately 4-fold following antibiotic therapy. The results indicate an association between GUD and increased blood HIV-1 RNA levels. Increased HIV-1 in semen was demonstrated in some men with GUD; such an increase could lead to increased transmission, thus complicating interpretation of the role of the genital ulcer itself in the infectiousness of HIV. Reasons for increased HIV RNA in semen in men with GUD remain to be determined.

Adult↗
PubMed1985

Partial purification of stable transcription complexes with cloned tRNA genes of Drosophila melanogaster.

The specific transcription of a cloned Drosophila melanogaster tRNAVal4 gene and a tRNASer7 gene by extracts from a homologous embryonic cell line showed lag periods of about 30 min before maximum rates were reached. This lag appeared to represent the time to form an active transcription complex. Thus, when extracts were incubated with template DNA for 30 min at 22 degrees C and stored in the cold, the subsequent transcription rate was linear with time and without a lag. After ultracentrifugation of a preincubated reaction mixture on a sucrose step gradient consisting of 20, 30, 40, and 60% shelves, about 40% of the transcription activity in the extract was found in the 40% shelf. This fraction formed almost exclusively RNA I, the unprocessed tRNA gene transcript, and transcription required only addition of ribonucleoside triphosphates. The rate of formation of RNA by the 40% sucrose fraction was linear against time, with no lag, and linear with the quantity of fraction. The yield of activity isolated on the gradient was directly proportional to the quantity of cloned gene in the preincubation mixture. At a limiting concentration of the gene in the preincubation mixture, the turnover number of the isolated complex was approximately 50 transcripts/gene/h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of fractions containing the complex still showed many bands, although the complex activity was greatly purified compared to the extract. From the sedimentation behavior of the isolated active transcription complex and from its stability and transcriptional properties, we conclude that the 40% sucrose fraction contains an active transcription complex containing a cloned tRNA gene, RNA polymerase III, and the accessory protein factors required for transcription.

Animals↗
PubMed1983

Developmental aspects of androgen-dependent mRNA from rat ventral prostate using cloned cDNA.

The androgen-dependence of two mRNAs from rat ventral prostate coding for a 20 000 and an 11 000 dalton translation product has been investigated using complementary DNA cloned in the bacterial plasmid PBR322. One of the cloned insert DNAs from a recombinant plasmid, C-27, arrests the in vitro translation of C2 (Peeters et al. (1980) J. Biol. Chem. 255, 7017-7023). The other cloned insert DNA arrests the translation of a glycoprotein 20 000 daltons in size, with unknown function. The quantities of mRNA coding for the 20 000 and 11 000 dalton translation product were determined by hybridization of 32P-labeled inserts to filter-bound total RNA or poly(A+)-mRNA. Castration caused a decline in both mRNAs of 250-fold over 8 days. Stimulation with androgen of 5-week castrates restored the mRNA levels to 17% of intact for the 20 000 dalton translation product and 31% of intact for the 11 000 dalton translation product. The quantity of the two mRNAs found in the lateral poly(A+)-mRNA was about 1/10 that of the ventral level and the mRNAs were not detectable in the dorsal prostate, seminal vesicle or human prostate poly(A+)-mRNA populations. RNA from the ventral prostates of animals 10-21 days old contained mature levels of complementary sequences, suggesting a form of developmental posttranscriptional regulation for synthesis of the polypeptides which are not synthesized in mature quantities at this stage of development (Heyns et al. (1978) Endocrinology 103, 1090-1095; Kistler et al. (1981) Proc. Natl. Acad. Sci. (U.S.A.) 78, 737-741).

Androgen-Binding Protein↗
PubMed1998

RNA quantitative analysis from fixed and paraffin-embedded tissues: membrane hybridization and capillary electrophoresis.

Fixed and paraffin-embedded tissues from pathology department archives are available for RNA expression analysis. We describe a general method for quantitation of specific RNA sequence extracted from single 6-8-micron human histological tissue sections cut from paraffin blocks. For each specific mRNA, the range of linear relationship between the log of the initial total RNA concentration and the log of the specific product after reverse transcription (RT)-PCR must be established. We usually perform RT with avian myeloblastosis virus (AMV)-RT, using specific antisense primers and a variable number of cycles of PCR amplification. The number of cycles must be adjusted within the range in which a linear relationship exists between the log of the amount of amplification product and the number of cycles. The quantity of specific product is standardized relative to beta-actin mRNA to normalize for the degree of RNA degradation, which can be quite different among samples. The amplification products were quantified by dot blot and 32P-labeled hybridization probe or by capillary electrophoresis with a laser-induced fluorescence detector. The intratest variation range was for the dot blot mean +/- 10% standard deviation (SD) and for the capillary electrophoresis mean +/- 3% SD.

Actins↗
PubMed2005

MicroInspector: a web tool for detection of miRNA binding sites in an RNA sequence.

Regulation of post-transcriptional gene expression by microRNAs (miRNA) has so far been validated for only a few mRNA targets. Based on the large number of miRNA genes and the possibility that one miRNA might influence gene expression of several targets simultaneously, the quantity of ribo-regulated genes is expected to be much higher. Here, we describe the web tool MicroInspector that will analyse a user-defined RNA sequence, which is typically an mRNA or a part of an mRNA, for the occurrence of binding sites for known and registered miRNAs. The program allows variation of temperature, the setting of energy values as well as the selection of different miRNA databases to identify miRNA-binding sites of different strength. MicroInspector could spot the correct sites for miRNA-interaction in known target mRNAs. Using other mRNAs, for which such an interaction has not yet been described, we discovered frequently potential miRNA binding sites of similar quality, which can now be analysed experimentally. The MicroInspector program is easy to use and does not require specific computer skills. The service can be accessed via the MicroInspector web server at http://www.imbb.forth.gr/microinspector.

Base Pairing↗
PubMed1969

Ultrastructural and biochemical changes in brown fat in cold-exposed rats.

During the first 3 days of exposure of rats to 5 degrees C, the nitrogen concentration of interscapular brown fat increased by 50% and remained at this elevated level for the duration of the 8-wk observation period, while the mass of tissue increased fourfold. The concentration of both DNA and RNA per unit nitrogen reached a maximum after 3 days, then declined; however, the total quantity of each continued to rise. The concentration of various respiratory enzymes decreased during the first few days and then increased, but at different rates. The morphological changes in mature brown fat cells during cold acclimation were observed to be: a reduction in fat droplet size during the first 3 days, followed by a gradual increase in size through 6 wk in the cold; a continual increase in the amount of intermitochondrial ground substance during the first 3 wk, with increased granularity and glycogen content after 1 wk; initial disappearance of glycogen between mitochondria, followed by the reappearance of a few isolated particles in the intermitochondrial ground substance after 1 wk in the cold; initial increase in the density of intramitochondrial matrix for the first 3-4 days, followed by a gradual return to the control density; loss in integrity of mitochondrial outer membranes during the first 4 days, followed by gradual but incomplete restoration; temporary loss of the dense material in lipid droplets during the first 24 hr, with return after 1 wk in the cold; and a 40% increase in mitochondrial diameter within 1 day, followed by a decrease in diameter within 1 wk to a constant value about 15% larger than the controls.

Adipose Tissue↗
PubMed1987

The role of the oncogene c-myc in sporadic large bowel cancer and familial polyposis coli.

The human myc gene is homologous to the v-myc gene, which was discovered in the avian oncogenic retrovirus MC29. Abnormally high expression of the cellular oncogenes is suspected to be involved in nonviral carcinogenesis. This article reviews the evidence that elevated expression of the human myc gene is involved in sporadic colon carcinoma and familial polyposis coli. The abundance of myc RNA and protein is frequently higher in colorectal cancer than in normal mucosa. The mechanism of this altered expression is obscure because the structure and quantity of myc DNA are very rarely disturbed. The COLO320 cell line is discussed as one unusual example of myc DNA rearrangement and amplification. There is a correlation between tumor-specific elevated myc RNA level and location in the bowel; tumors distal to the transverse colon are more likely to have elevated myc expression. The site distribution of unselected colorectal malignancies with an elevated myc RNA level is similar to the site distribution of familial polyposis coli tumors. With this observation it is suggested that elevated myc RNA may be a marker of a distinct type of colorectal cancer that involves the same genetic events as precede familial polyposis coli cancer.

Adenomatous Polyposis Coli↗
PubMed1975

Histological and histochemical study on the ependyma of Bradypus tridactylus.

Histological and histochemical aspects of the whole encephalic ventricular system of eight specimens of Bradypus tridactylus were studied. After anesthesia and perfusion, the encephalons were obtained by craniotomy. Transverse serial sections of the encephalon, stained according to Azan (Heidenhain's method) or Kluver-Barrera for nerve cells and myelinated nerve fibers; silver impregnation was carried out according to Cajal-De Castro's or Palmgren's methods. The following histochemical reactions were used: PAS (McManus), metachromasia, acid phosphatase (Gomori), Brachet's and Gomori's trichromic reaction (modified by Bargmann for neurosecretion). Histologically, different characteristics of the ependymal cells in different areas were observed, which would be related to functional peculiarities of each area of the encephalic ventricles. The ependymal cells showed discrete apical basophilia due to the presence of RNA which disappears after treatment with crystalline ribonuclease. The PAS reaction indicated the presence of a small quantity of PAS-positive substances in the apical zone of the ependymal cells and the subependymal tissue. These substances disappeared after the salivary amylase test, indicating the presence of glycogen. The acid phosphatase reaction was negative.

Aminosalicylic Acids↗
PubMed1995

Variable numbers of BCR-ABL transcripts persist in CML patients who achieve complete cytogenetic remission with interferon-alpha.

A substantial minority of patients with chronic myeloid leukaemia (CML) achieve a complete response to treatment with interferon-alpha (IFN-alpha), defined as the disappearance of Philadelphia chromosome positive metaphases or, for patients who are Philadelphia chromosome negative but BCR-ABL positive, the disappearance of the leukaemic clone as assayed by Southern blot. We have measured the levels of BCR-ABL transcripts in 20 such patients by quantitative PCR. Results were standardized for both quality and quantity of cDNA by quantification of ABL as an internal control. All 20 patients had evidence of residual disease; the median number of transcripts was 750/micrograms RNA (range 10-22,000) and the median BCR-ABL/ABL ratio was 0.17% (range 0.0008-3.6%). Our findings show that CML has not been eradicated in any patient and that the quantity of residual disease in complete responders may vary by as much as four orders of magnitude.

Adult↗
PubMed2005

Assessment of a new RNA stabilizing reagent (Tempus Blood RNA) for minimal residual disease in onco-hematology using the EAC protocol.

Standardization efforts of real time quantitative PCR (RQ-PCR), such as the Europe Against Cancer (EAC) protocol, are actually essential. However, all the stages of the preanalytical phase (blood collection, conservation and procedures of cellular separation) which influence the ex vivo expression of many genes are not controlled. Various kits for stabilizing the RNA at the time of blood collection were developed: PAXgene Blood kit (Qiagen) and the new Tempus Blood RNA kit (Applied Biosystems). PAXgene Blood was already validated to monitor minimal residual disease (MRD) in onco-hematologic pathologies. In order to evaluate the Tempus Blood RNA kit, it was compared to unstabilized EDTA blood/Ficoll/TRIzol protocol and PAXgene Blood kit. The RNAs extracted by the different methods were assessed for quantity, quality and detection of control genes (GUS and ABL) and fusion gene transcripts BCR-ABL and TEL-AML1 by RQ-PCR. Our study shows that using the EAC protocol, the new Tempus Blood RNA kit allows the detection of fusion gene transcript with the same sensitivity as the two other protocols. Altogether, our data suggest the possible use of such a technology for MRD follow-up in myeloproliferative diseases and acute leukemias. So, further multicentric studies on leukemic patients should be performed to validate this technique in these applications.

Cell Line, Tumor↗
PubMed1997

RNA-protein interactions: involvement of NS3, NS5, and 3' noncoding regions of Japanese encephalitis virus genomic RNA.

The mechanism of replication of the flavivirus Japanese encephalitis virus (JEV) is not well known. The structures at the 3' end of the viral genome are highly conserved among divergent flaviviruses, suggesting that they may function as cis-acting signals for RNA replication and, as such, might specifically bind to cellular or viral proteins. UV cross-linking experiments were performed to identify the proteins that bind with the JEV plus-strand 3' noncoding region (NCR). Two proteins, p71 and p110, from JEV-infected but not from uninfected cell extracts were shown to bind specifically to the plus-strand 3' NCR. The quantities of these binding proteins increased during the course of JEV infection and correlated with the levels of JEV RNA synthesis in cell extracts. UV cross-linking coupled with Western blot and immunoprecipitation analysis showed that the p110 and p71 proteins were JEV NS5 and NS3, respectively, which are proposed as components of the RNA replicase. The putative stem-loop structure present within the plus-strand 3' NCR was required for the binding of these proteins. Furthermore, both proteins could interact with each other and form a protein-protein complex in vivo. These findings suggest that the 3' NCR of JEV genomic RNA may form a replication complex together with NS3 and NS5; this complex may be involved in JEV minus-strand RNA synthesis.

Binding Sites↗
PubMed1976

Comparative adduct formation of 4-aminobiphenyl and 2-aminofluorene derivatives with macromolecules of isolated liver parenchymal cells.

Isolated parenchymal cells of rat liver have been used in a study of the metabolic activation of derivatives of the carcinogens 4-aminobiphenyl and 2-aminofluorene. The formation of adducts of these compounds with cellular RNA and protein has been taken as evidence of their transformation to metabolites that are capable of spontaneous reaction with tissue macromolecules. The hydroxamic acid N-hydroxy-N-4-acetylaminobiphenyl was bound to RNA to a greater extent than were the amino-, hydroxylamino-, nitroso-, nitro-, acetylamino-, or azoxybiphenyl derivatives. RNA adducts of the hydroxamic acid retained little of the acetyl group. The structural requirements for binding and the nature of the bound derivatives are consistent with the activation of N-hydroxy-N-4-acetylaminobiphenyl by N leads to O acyltransfer. Approximately equal quantities of 4-nitrosobiphenyl and the hydroxamic acid were bound to protein, but far less of the nitroso derivative was incorporated into RNA. Adduct formation of N-hydroxy-N-2-acetylaminofluorene with RNA occurred with retention of the acetyl group and was dependent on the concentration of sulfate in the media. Consequently, reaction of the fluorenyl derivative with RNA probably resulted from conjugation of the hydroxamic acid with sulfate.

Acetates↗
PubMed1974

Purification of avian myeloblastosis virus DNA polymerase by affinity chromatography on polycytidylate-agarose.

Polycytidylic acid [poly(rC)] covalently linked to cyanogen bromide-activated agarose is an effective affinity matrix for the RNA-dependent DNA polymerase from avian myeloblastosis virus. Poly(rC)-agarose is capable of binding large quantities of avian myeloblastosis DNA polymerase, which is then eluted by using a linear KCl gradient of increasing concentration. The DNA polymerase isolated from crude, detergent-disrupted virions by a single pass through columns of poly(rC)-agarose appears nearly homogeneous (approximately 90% pure) as determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Complete recovery of input enzymatic activity was obtained. Results suggest that polyribonucleotide columns may provide a high-yield, rapid method for the purification of oncornaviral DNA polymerase.

Avian Leukosis Virus↗
PubMed1975

Use of a specific probe for ovalbumin messenger RNA to quantitate estrogen-induced gene transcripts.

DNA complementary to purified ovalbumin messenger RNA (cDNA ov) was synthesized in vitro using RNA-directed DNA olymerase from avian myeloblastosis virus. This cDNAov was then employed in hybridization assays to determine the effect of estrogen on the number of ovalbumin mRNA (MRNAov) molecules per tubular gland cell of the chick oviduct. The changes in mRNAov were measured in immature chicks during primary stimulation, after hormone withdrawal and again following secondary stimulation of the chick oviduct with estrogen. The number of mRNAov per tubular gland cell was also determined for egg-laving hen. Daily estrogen administration to the immature chick resulted in growth of the oviduct, differentiation of epithelial cells to tubular glands, and a corresponding increase in the concentration of mRNAov in the tubular gland cell from essentially zero before estrogen administration to 48,000 molecules per cell after 18 days of estrogen treatment. Upon withdrawal of estrogen from the chick, the mRNAov concentration decreased to a level of 0-10 molecules/tubular gland cell after 12 days. Readministration of a single dose of estrogen to these chicks resulted in a dramatic and rapid increase in the concentration of mRNAov. Within 30 min, the mRNAov concentration approximately doubled and by 29 hr the tubular gland cell concentration had reached 17,000 molecules. The initial transcription rate for the ovalbumin gene was 12 mRNAov molecules/min. With these data, we have calculated that the half-life of the ovalbumin messinger RNA should be on the order of 40-60 hr and that the steady-state concentration of mRNAov per tubular gland cell was 50,000 molecules. Similarly, each messenger RNA molecule was translated approximately 50,000 times during its lifetime in order to effect the necessary quantity of ovalbumin required for egg production. These data substantiate the hypothisis that estrogen exerts its primary action at the level of transcription to effect the synthesis of nascene mRNA molecules which in turn code for synthesis of hormone-induced proteins.

Animals↗
PubMed2006

Intracellular localization and content of YB-1 protein in multidrug resistant tumor cells.

The multifunctional mammalian protein YB-1 is a member of the large DNA- and RNA-binding protein family with an evolutionarily ancient cold-shock domain. YB-1 is involved in multiple DNA- and mRNA-dependent events and regulates gene expression at various levels. It can be found both in the nucleus and the cytoplasm. Bound to DNA in the cell nucleus, YB-1 functions as a transcription factor interacting with inverted CCAAT-box (Y-box) in promoters and enhancers of multiple genes. In particular, YB-1 regulates activity of the multidrug resistance (MDR) genes MDR1 and LRP. In tumors, YB-1 has been suggested to be an early and global marker of MDR. In this study, we compared amounts of YB-1 mRNAs and intracellular localization of YB-1 protein in six pairs of drug sensitive and drug resistant sublines of diverse tumors. We have shown that neither great increase in the level of YB-1 mRNA nor substantial increase in the number of cells with nuclear localization of YB-1 are obligatory traits of drug resistant tumor cell populations. However, the cells with highest amounts of YB-1 mRNA also demonstrated increased quantities of MDR1, MRP1, BCRP, and LRP mRNAs encoding different MDR proteins. Transfection of two different populations of drug-sensitive cells with YB-1 cDNA led to increase in the amount of YB-1 mRNA. The quantities of MRP1 and LRP mRNAs increased in both populations. Introduction of YB-1 small hairpin RNA (shRNA) resulted in decreased amounts of YB-1 mRNA, as well as MRP1, LRP, and MDR1 mRNAs (in three different cell lines). Our data suggest that although YB-1 regulates several MDR genes, it could not be regarded as a global marker of already formed drug resistant tumor cell populations. It is most likely that at the first steps of MDR development YB-1 activity is necessary for propagation of resistant cell populations rather than for maintenance of drug resistance.

Amino Acid Sequence↗