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PubMed1996

Synergistic enhancement of both initiation and elongation by acidic transcription activation domains.

The effects of activation domain synergy on transcription initiation and elongation have been examined utilizing a system that permits the targeting of a defined number of activation modules to promoter DNA. As predicted, incremental increases in targeted activation potential were found to result in corresponding increases in transcription initiation. Surprisingly, however, transcriptional processivity, and hence mRNA synthesis, required a threshold level of activation domain synergy that exceeded the level required for at least modest levels of transcription initiation. The degree to which transcriptional processivity was enhanced was shown to depend on the quantity of activation modules targeted to the promoter DNA, rather than the quality. While the RNA-sequence specific HIV-1 Tat trans-activator was also shown to enhance processivity in this assay system, Tat differed from DNA-sequence specific activation domains in exerting a more dramatic effect on the efficiency of transcript elongation.

Animals↗
PubMed1995

[Effects of some physical and chemical factors on both human hair DNA and results of sex determination by PCR].

The present study aims at observation of the effects of some physical and chemical factors on both human hair DNA and the results of sex determination by PCR. After the hairs were treated with varying pH solutions, the quantity of the DNA extracted was decreased. High-molecular-weight (HMW) DNA was determined in the hairs treated with pH 1, 3, 5 and 7 solutions for 2 hours, but no DNA was demonstrated by agarose gel electrophoresis in the hairs treated with pH 1 and 3 solution for 24 hours, and the quantity of DNA was decreased in the hairs treated with pH 5 for 24 hours. The quantity of DNA was decreased in the hairs treated with pH 9 for 2 hours and no DNA was demonstrated for 24 hours. No DNA was found in the hairs treated with pH 11 for 2 or 24 hours. No changes of DNA were observed in hairs treated with 75% ethanol or methanol for 2 or 24 hours and exposed to UV light for 24 hours. No DNA but RNA was observed in the hairs treated with 10% formalin (pH 7 or pH 5) for 2 or 24 hours. The quantity of DNA was decreased obviously in the hairs treated in 100 degrees C water for 5 or 10 minutes. The quantity of DNA was not decreased in the hairs heated at 50 degrees C for 24 hours, and only little decrease was noted at 100 degrees C for 24 hours. Both fragments of PCR products were obtained in most hairs except the hairs treated with pH 11 solution for 24 hours. Although no DNA was observed in some treated hairs, specific bands were detected after PCR. PCR is a very sensitive and specific technique of DNA amplification, which can be used for sex determination in forensic medicine.

Base Sequence↗
PubMed1978

Asymmetric transcription during post-germinative development of Bacillus subtilis spores.

The relative transcription from L and H strands of Bacillus subtilis DNA during consecutive stages of spore outgrowth was determined and compared to the transcription pattern during log-phase growth of vegetative cells. Pulses of [3H] uridine were administered during early, middle and late outgrowth phases of germination and the RNAs isolated. The asymmetry ratio of H/L as determined by hybridization at saturating RNA/DNA inputs showed a gradual decrease. During the period studied (10-90 and 90-160 min post-induction), about 50 and 35%, respectively, of the radioactive RNA consisted of ribosomal RNA transcripts. The decrease in the H/L asymmetry ratio was due predominantly to the appearance and accumulation of L strand transcripts and not to either changes in the quantity of H strand transcripts nor to fluctuation in the rate of rRNA synthesis.

Bacillus subtilis↗
PubMed1996

Editing of GluR2 RNA in the gerbil hippocampus after global cerebral ischemia.

Postischemic delayed neuronal death (DND) in CA1 of the gerbil hippocampus is thought to be caused by an abnormal increase of Ca2+ influx into the cell, mediated by excessive activation of glutamate receptors. One subtype of glutamate receptors, the AMPA receptor, is not permeable to calcium ions as long as an edited form of its GluR2 subunit is present. It is possible that global ischemia interferes with the posttranscriptional editing of the GluR2 mRNA and thus leads to calcium influx via the AMPA receptor. In order to test this hypothesis, we examined the extent of GluR2 RNA editing in CA1 and CA3 microdissected from gerbil hippocampus after 5 min of global ischemia and various recirculation intervals. At each interval tested, quality and quantity of mRNA editing in the vulnerable CA1 region were the same as in CA3. Furthermore, postischemic mRNA editing in both hippocampal regions was indistinguishable from editing in untreated control animals. Our results clearly demonstrate that global ischemia does not cause impairment of GluR2 RNA editing, which is thus not responsible for the abnormal calcium permeability of the postischemic cell membrane.

Animals↗
PubMed1999

Cyclin B1 transcript quantitation over the maternal to zygotic transition in both in vivo- and in vitro-derived 4-cell porcine embryos.

Using reverse transcription-competitive polymerase chain reaction (RT-cPCR), the quantity of cyclin B1 transcript present over the maternal to zygotic transition was determined for both in vivo- and in vitro-derived 4-cell porcine embryos. After poly(A) RNA isolation, RT-cPCR was performed on single embryos using an introduced, truncated cyclin B1 DNA competitor. Visualization of embryonic cyclin B1 cDNA and competitor for each reaction allowed a ratio to be formed for use in transcript quantity calculations when compared to cPCR standards. Analysis of in vivo- and in vitro-derived control embryos revealed a decline in cyclin B1 transcripts from 5 to 33 h post-4-cell cleavage (P4CC). The quantity of cyclin B1 for the in vivo-derived embryos at 5 and 33 h P4CC was 11.26 and 4.54 attomol/embryo, respectively (P < 0.03), while the in vitro-derived embryos had 20.18 and 7.52 attomol/embryo, respectively (P < 0.03). Treatment with alpha-amanitin from 5, 10, 18, or 25 h P4CC to 33 h P4CC resulted in cyclin B1 quantities that did not differ from those in the 33-h control embryos, irrespective of time spent in the inhibitor. These findings suggest that maternal cyclin B1 transcript degradation occurred over the 4-cell stage with no detectable embryonic cyclin B1 transcripts produced.

Amanitins↗
PubMed1991

Production of the neuronal growth-associated protein GAP-43 in a bacterial expression system.

GAP-43, a major protein of neuronal growth cones and certain presynaptic terminals, is a candidate for important functions in both axon growth and synaptic plasticity. To facilitate studies that may elucidate these functions, we have efficiently generated large quantities of GAP-43 by introducing a GAP-43 cDNA into a bacterial expression system driven by T7-RNA polymerase. Two constructs were expressed in this system: one (pT7Ava-GAP) produces a fusion protein in which the first 16 amino acids of GAP-43 are replaced by 11 amino acids of the phage T7 capsid protein; the other (pT7FL-GAP) produces full length GAP-43. After the bacteria were lysed, both products were soluble, and could be efficiently purified by HPLC chromatography on a C4 reversed-phase column. One liter of bacterial culture yielded 50 mg of purified fusion protein or 10 mg of complete GAP-43. When it was incubated with protein kinase C, the fusion protein was phosphorylated at the same single site (serine 41) that is phosphorylated in cultured neurons. The ability to produce large quantities of GAP-43 by this procedure should expedite future studies investigating its structure, posttranslational modification, and function.

Amino Acid Sequence↗
PubMed1987

Defective HLA class II expression in a regulatory mutant is partially complemented by activated ras oncogenes.

The human B-cell line RJ2.2.5, derived by mutagenesis from a Burkitt lymphoma cell line and selected for loss of HLA class II antigen expression, was infected with recombinant retroviruses containing either the Harvey murine sarcoma virus oncogene v-Ha-ras or the human neuroblastoma homolog NRAS. Both activated ras genes partially complemented the regulatory defect in RJ2.2.5 and specifically increased the expression of the DR and DQ subsets of HLA class II genes. Blot-hybridization analysis and RNase mapping indicated that HLA-DQ alpha-chain mRNA in the infected cell lines was increased to a level at least 50% that of the parent B-cell line, Raji. The levels of HLA-DR and -DQ beta-chain RNA also were increased but to a lesser extent. In contrast, we detected no effect of ras on the quantities of other class II, class I, or invariant-chain mRNAs. Fluorescence-activated cell sorter analysis with antibodies recognizing HLA-DR, -DQ, and class I antigens supported these observations. Enhancement of HLA class II gene expression by ras genes may have important implications for regulation of the immune system in response to transformation.

Antigens, Surface↗
PubMed1995

Hepatitis C virus in alcoholic patients with and without clinically apparent liver disease.

A high prevalence of antibodies to the hepatitis C virus (anti-HCV) has been demonstrated among patients with alcoholic liver disease, whereas the prevalence of HCV viremia in these patients remains uncertain. The aims of this study were to determine the prevalence of anti-HCV in alcoholic patients both with and without clinically apparent liver disease and to determine the presence of HCV RNA in those patients who tested positive for anti-HCV by RIBA II (Chiron Corporation, Emeryville, CA). One hundred male patients consecutively admitted to an alcoholic rehabilitation program were included. Group 1 was comprised of 40 patients with clinically apparent liver disease. Group 2 was comprised of 60 patients without clinically apparent liver disease. Anti-HCV was performed by a second-generation ELISA assay and confirmed by RIBA II. HCV RNA was performed by Quantiplex assay (Chiron Corporation) and a nested reverse transcriptase-polymerase chain reaction. No significant differences were found between the two groups with regards to age, quantity and duration of alcohol intake, or accepted risk factors for HCV. The overall prevalence of anti-HCV in our patients was 23%, with 43% of these in group 1 and 10% in group 2. HCV RNA tested positive in 94% of the anti-HCV-positive patients in group 1 and in 67% of the anti-HCV-positive patients in group 2. These data suggest that HCV infection is an important cofactor in the pathogenesis of liver disease among alcoholic patients.

Adult↗
PubMed1998

Analysis of putative RNase sensitivity and protease insensitivity of demethylation activity in extracts from rat myoblasts.

The mechanism for demethylation of DNA in rat myoblasts has recently been studied using a new in vitro system that monitors demethylation in whole cell extracts. Previous investigations using this system had indicated that demethylation is resistant to conditions that are normally assumed to denature or digest proteins. Remarkably, it was reported that the activity appeared to be sensitive to the action of ribonuclease, suggesting a role for RNA in the demethylation of DNA. This manuscript reports that, upon further purification of the extract, demethylation activity has properties that are different. When subjected to more rigorous procedures for digestion of proteins, the demethylase activity disappears. Furthermore, RNase sensitivity of the extract disappears when a quantity of unmethylated competitor DNA is added to the reaction mix or when extracts treated with RNase are subsequently treated with protease. Although a role for RNA cannot be completely discounted, it is unlikely that this demethyl-ation reaction involves RNA cofactors or ribozyme components. These results have important implications for the mechanism of DNA demethylation and they exemplify the potential pitfalls of experiments in which new biological roles for RNA are evaluated using RNase sensitivity experiments.

Animals↗
PubMed1979

Ileal resection potentiates 1,2-dimethylhydrazine-induced colonic carcinogenesis.

The effect of colonic hyperplasia produced by resection of the distal third of the small bowel (DSBR) on the development of chemical carcinogenesis of the colon by dimethylhydrazine (DMH) was tested in rats. For the most part the amounts of RNA and DNA in the small bowel were the same with the combined treatments as with either one alone; quantities of nucleic acids tended to increase only in transverse and distal colon. After 37 weeks the number of neoplasms per rat was increased six-fold by combining DSBR with DMH. Neoplasms were spread throughout the colon after combined treatment as opposed to the ascending colong after DMH alone. Postresectional hyperplasia appears to increase the incidence and distribution of colon tumors.

Animals↗
PubMed1985

The fate of heterogeneous nuclear ribonucleoprotein complexes during mitosis.

Using immunochemical techniques, we have examined the macromolecular state of association of the major heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins in mitotic HeLa cells. We find that these proteins are not free but are associated with high-molecular-weight RNA in the form of particles that sediment as a broad band between 80 and 200 S. We have termed these complexes MhnRNP for mitotic hnRNP protein-containing particles. Their quantity, composition, sedimentation coefficients, buoyant density, and sensitivity to dissociating conditions suggest that they are closely related to the hnRNP complexes of interphase cells and may represent hnRNP complexes containing unprocessed or partially processed heterogeneous nuclear RNA that have been released into the cytoplasm during mitosis. Exogenously added RNA does not associate with the MhnRNP nor does it compete for the major MhnRNP proteins. The MhnRNP remain distinct from other ribonucleoprotein complexes and do not associate with ribosomes even though these structures are not separated by a nuclear envelope during mitosis.

Enzyme-Linked Immunosorbent Assay↗
PubMed1989

Diminished expression of the T cell receptor on the expanded lymphocyte population in MRL/Mp-lpr/lpr mice.

MRL mice homozygous for the recessive lpr gene develop an accelerated autoimmune syndrome and massive lymphadenopathy. Because the function of the expanded lymph node population is unclear, we have studied the subunits of the T cell receptor for antigen (TcR). DNA and RNA were prepared from MRL/Mp-lpr/lpr (lpr) and congenic MRL/Mp(-)+/+ (+/+) mice by standard techniques and studied by Southern blot, northern blot, and dot blot analysis using the cDNAs TT11, specific for the TcR alpha chain; 86T5, specific for the TcR beta chain; and T3 delta; specific for the subunit of the T3 molecule. Surface protein was immunoprecipitated with antisera 8177, which recognizes TcR framework determinants, and resolved by diagonal SDS-PAGE. FACS analysis was performed with a monoclonal antibody to murine T3, and with the KJ16-133 and F23.1 monoclonal antibodies, which recognize determinants encoded by the V beta 8 subfamily of beta chain variable region genes. When compared with +/+ controls, surface TcR density as detected by immunofluorescence using all three antibodies was significantly diminished on lpr spleen and lymph node cells, as well as on lpr lymph node cells which had been depleted of L3T4+ and Ly2+ cells by negative selection. There appeared, however, to be selective expression of the genes encoding the epitopes binding F23.1. Southern blot analysis of DNA showed polyclonal rearrangements of the TcR beta chain genes. There were increased alpha, beta, and T3 delta RNA transcripts in the double negative lymph node cells. The paradoxical decrease in TcR surface expression in the setting of large quantities of full length transcript is yet to be explained.

Animals↗
PubMed1973

Studies of nondefective adenovirus 2-simian virus 40 hybrid viruses. VII. Characterization of the simian virus 40 RNA species induced by five nondefective hybrid viruses.

Five nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses have been isolated and found to contain segments of SV40 DNA covalently linked to Ad2 DNA. The quantity of SV40 DNA present is a stable characteristic of each hybrid virus, and varies from less than 5% (in Ad2(+)ND(3)) to more than 30% (in Ad2(+)ND(4)) of the SV40 genome. We have characterized the SV40 portions of these hybrids by relating the SV40-specific RNA sequences transcribed in cells infected with each hybrid virus to those transcribed in cells infected with each of the other hybrid viruses and with SV40 itself. RNA-DNA hybridization-competition experiments indicate that the number of unique SV40 RNA sequences transcribed in infected cells is proportional to the size of the SV40 DNA segment contained within each hybrid and, in the case of the three hybrids which induce detectable SV40-specific antigens, to the number of SV40 antigens induced. Furthermore, the SV40-specific RNA sequences transcribed from any one of the hybrids are completely represented in the RNA transcribed from all other hybrids with longer SV40 segments. Thus, the SV40 DNA regions in the five hybrid viruses appear to contain some nucleotide sequences in common. The SV40-specific RNA transcribed from Ad2(+)ND(4), the hybrid containing the largest SV40 segment, is qualitatively similar to the SV40-specific RNA transcribed early (i.e., prior to viral DNA replication) in SV40 lytic infection. Thus, it appears that no significant amount of late SV40 DNA is transcribed during infection by any of the five nondefective Ad2-SV40 hybrid viruses.

Adenoviridae↗
PubMed1992

Translation of potato virus S RNA in vitro: evidence of protein processing.

RNA from potato virus S (PVS), a member of the carlavirus group, was translated in vitro in rabbit reticulocyte lysate. Time-course experiments revealed the largest product of Mr 190 kD, decreasing in intensity after 60-min incubations, correlating with the accumulation of a 150-kD peptide. This apparent processing could be blocked by the addition of the amino-acid analogues p-fluorophenylalanine and L-canavanine for phenylalanine and arginine, respectively. L-canavanine also appeared to specifically reduce the quantity of PVS (34 kD) coat protein, concomitant with the synthesis of a 36-kD peptide. Sucrose gradient-fractionated genomic RNA directed the synthesis of predominantly 190-kD peptides that appeared not to be processed in the absence of small molecular weight (subgenomic) RNA products.

Alphavirus↗
PubMed1994

The biological effects of antisense N-myc expression in human neuroblastoma.

Although N-myc amplification and overexpression are believed to play an important role in determining the clinical behavior of neuroblastoma (NB), the exact function of N-myc in NB cell growth and differentiation remains unknown. To better understand the function of N-myc, an established human NB cell line was transfected with N-myc antisense (AS) complementary DNA in an effort to down-regulate N-myc gene expression. Five clones expressing AS N-myc RNA have been maintained in culture for over 2 years. Compared to control cells, a 30-69% decrease in the quantity of N-myc protein was demonstrated by Western blot analysis in 4 of the 5 AS clones. All 5 of the AS clones exhibited a 50-75% decrease in colony formation in soft agar assays compared to control cells. In addition, all 5 AS clones expressed a 3.2-kilobase protein kinase C-alpha transcript, whereas this message was not detected by Northern blot analysis in any of the control clones. These results suggest that N-myc may play an important role in NB cell growth and that antisense N-myc expression is associated with an induction of protein kinase C-alpha RNA expression. Further characterization of the AS clones may provide insight into the function of N-myc and may thus lead to a better understanding of the role that N-myc plays in determining the clinical behavior of this childhood neoplasm.

Blotting, Northern↗
PubMed1998

Measurement of feline cytokine gene expression by quantitative-competitive RT-PCR.

We have developed a method to quantitate feline cytokine gene expression using competitive RT-PCR. Feline cytokine specific primers were developed that encompass an intron, thus allowing differentiation of cDNA vs. genomic DNA amplification products. The PCR products of the primers were verified by sequencing and Southern blot analysis. For quantitation, a non-homologous RNA competitor was created for each cytokine of interest. The competitor was designed to yield an RT-PCR product 10-20% larger than the native sequence, thereby allowing differentiation of the two products by electrophoresis on an agarose gel. Both competitor and native sequences used the same primer sequences for RT (oligo dT) and PCR (cytokine specific). The amplification efficiency of the competitor and native sequence was shown to be identical which allowed comparison at any point during the amplification, including the plateau phase. The quantity of starting cytokine mRNA was determined by interpolation from a standard curve. As little as 1 microgram of total cellular RNA was required per cytokine determination. The assay can routinely quantify as few as 1000 copies of template and spans a range of up to 4 log.

Animals↗
PubMed1997

Serum HCV-RNA and liver histologic findings in patients with long-term normal transaminases.

In this study we aimed to correlate liver histology and the presence of hepatitis C virus (HCV) viremia, genotype, and quantity of HCV genome in 19 positive and 11 RIBA II indeterminate patients presenting persistently normal ALT values over 24 months before biopsy. In addition, after biopsy serum ALT values were monitored monthly for a mean follow-up period of 24.8 months, after which patients were reevaluated for RIBA II and the presence of viremia. Sixteen patients (53%) were serum HCV-RNA-positive; 13 of them (68%) were confirmed positive and 3 (27%) indeterminate on RIBA II. Histology of the HCV-RNA-positive patients showed eight cases of CPH (one case of genotype 1a; four cases type 1b; three cases type 2), six cases of CAH (three cases type 1b, three cases type 2), one case of CLH (type not determined), and one case of normal liver (NL) (type 1b). Histology of the HCV-RNA-negative patients showed four cases of CPH, one case of CAH, two cases of CLH, and seven cases of NL. During the follow-up period nine patients (30%) presented slight increases in ALT values (< 2 x N), and in particular, flares of ALT were observed four times in the CAH and five times in the CPH patients, who were all viremic, but never in the NL subjects. These results indicate that subjects positive on RIBA II, but with persistently normal ALT values, had a high probability of being serum HCV-RNA-positive and that almost all these viremic subjects presented histologic signs of liver disease. In contrast, RIBA II indeterminate subjects had a moderate probability of being HCV-RNA-positive, but a number of these may present signs of liver disease. In both cases there was no association with genotype or HCV-RNA serum levels. The other nonviremic cases included subjects with hepatic changes going toward resolution or with normal liver in whom hepatic biopsy can be avoided. Only one case was a true carrier since he was viremic with normal liver and persistently normal ALT values.

Adult↗
PubMed1991

Molecular characterization of hog cholera virus.

An efficient tissue culture system was established which allowed to obtain substantial quantities of hog cholera virus (HCV) from the cell free tissue culture supernatant. After preparation of viral RNA and cDNA synthesis, the complete HCV genome was cloned and sequenced. Comparison with published BVDV sequences revealed a surprisingly high homology between HCV and BVDV at both the nucleotide and the amino acid level. In addition host cellular sequences were identified in BVDV genomes. The genomic localization of HCV glycoproteins was determined by the use of sequence specific antisera directed against bacterial fusion proteins. The order on the HCV genome was determined as follows: N-gp44/48-gp33-gp55-C. HCV gp33 and HCV gp55 were shown to be intracellularly linked by disulfide bridges. A cDNA fragment covering the genomic region that encodes the structural proteins of HCV was inserted into a vaccinia recombination vector. Expression studies with vaccinia/HCV recombinants led to identification of HCV specific glycoproteins which migrated on sodium dodecyl sulfate-gels identically to glycoproteins precipitated from HCV-infected cells. The vaccinia virus/HCV recombinant that expressed all four structural proteins induced virus-neutralizing antibodies in mice and swine. After immunization of pigs with this recombinant virus, full protection against a lethal challenge with HCV was achieved. A construct that lacked most of the HCV gp55 gene failed to induce neutralizing antibodies but induced protective immunity.

Animals↗