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PubMed2002

Parameters of human hepatitis delta virus genome replication: the quantity, quality, and intracellular distribution of viral proteins and RNA.

Assembly of hepatitis delta virus (HDV) in infected human hepatocytes involves association of the 1,679- nucleotide single-stranded genomic RNA (deltaRNA) with multiple copies of both small and large forms of the delta protein (deltaAg) to form a ribonucleoprotein particle which in turn interacts with envelope proteins of the natural helper virus, hepatitis B virus. Subsequently, for initiation of a new round of replication, the amount of small deltaAg within the assembled HDV particle is both necessary and sufficient. Quantitative assays were used in order to better understand just how much deltaAg is needed. The molar ratio of deltaAg species to genomic deltaRNA in assembled HDV particles was approximately 200. Next, this ratio was determined for cells under several different experimental situations in which HDV genome replication was occurring. These included replication in woodchuck liver and also in mouse liver and skeletal muscle, as well as replication in stably and transiently transfected cultured human hepatoblastoma cells. Surprisingly, in almost all these situations the molar ratios were comparable to that observed for HDV particles. This was true for different times after the initiation of replication and was independent of whether or not virus assembly was occurring. Cell fractionation combined with quantitative assays was used to test whether the majority of deltaAg and deltaRNA were colocalized during HDV replication in transfected cells. The cytoplasmic fraction contained the majority of deltaAg and genomic deltaRNA. Finally, the quality of deltaAg and deltaRNA, especially at relatively late times after the initiation of replication, was examined by using reverse transcription-PCR, cloning, and sequencing through the entire deltaAg open reading frame. When virus assembly and spread were not possible, 20% or less of the predicted deltaAg would have been able to support HDV replication. In summary, an examination of the quantity, quality and intracellular distribution of deltaAg and deltaRNA in several different experimental systems has provided a better understanding of the parameters associated with the initiation, maintenance, and ultimate decline of HDV genome replication.

Animals↗

PubMed2001

Expression profiling of ductal carcinoma in situ by laser capture microdissection and high-density oligonucleotide arrays.

Gene expression profiling through the use of nucleic acid arrays is a powerful method for the molecular classification of human neoplasms. Laser capture microdissection is an equally useful technique to selectively isolate defined cell populations from heterogeneous histological tissue sections. In this report, we demonstrate how a modest use of laser capture microdissection is sufficient to isolate nanogram quantities of high-quality RNA. Together with the use of several internal standards and microcapillary electrophoresis of input RNA, two rounds of linear molecular amplification have been used to generate sufficient quantities of labeled target for hybridization to high-density oligonucleotide expression arrays. Results demonstrate that the technique is reproducible, generates only modest biasing of the original transcript population, and is comparable to the sensitivity achieved with standard methodology. Using this approach, we have compared the expression profiles of nonmalignant human breast epithelium and adjacent ductal carcinoma in situ lesions from breast cancer patients. Several genes, previously implicated in human breast cancer progression, demonstrate differential expression among the microdissected cell populations.

Breast↗

PubMed1988

Biotinylated and radioactive cDNA probes in the detection by hybridization of bovine enteric coronavirus.

cDNA, synthesized on bovine coronavirus (BCV) genomic RNA templates, could be used to detect very small quantities (i.e. 1 pg) of viral RNA by hybridization with either radioisotopic-labelled or biotinylated recombinant plasmids. Virus was optimally attached to nitrocellulose membranes when spotted in 1 x SSC, whereas 20 x SSC was superior for viral RNA. Denaturation and RNA fixation of both RNA, still encapsidated in virus particles and isolated genomic RNA, was achieved by baking of the blots in vacuum. Virus detection in the supernatant of infected HRT-18 cells was feasible, but improved significantly after proteinase K treatment. No homology was observed between virus cDNA with either plasmid DNA or nucleic acid isolated from non-infected HRT-18 cells. Hybridization with radioisotopic-labelled probes in higher formamide concentrations (up to 60%) increased the detection signals, possibly by reducing reassociation of the probe. Significant detection amplification (30-50 times) was achieved in the case of biotinylated probes by stimulation of hyperpolymer formation on already hybridized target sequences, by additional hybridization with biotinylated pUC-19. A detection amplification was also obtained when hybridization was done with two probes (pBC-52 and pBC-247), containing non-overlapping viral sequences. Although the detectability was surpassed by biotinylated probes, sensitivity was superior in radioisotopic virus detection.

Animals↗

PubMed2006

Influence of storage temperature on the stability of HIV-1 RNA and HSV-2 DNA in cervicovaginal secretions collected by vaginal washing.

Variability in the handling of samples of genital secretions prior to quantitation of HIV-1 RNA and HSV DNA may profoundly affect both the detection and quantitation of these nucleic acids. Over 144 h, we evaluated, the influence of storage temperature (4 degrees C, 20 degrees C, 30 degrees C) on the quantity of HIV-1 RNA and HSV-2 DNA in HIV and HSV negative cervicovaginal lavage pools spiked with known amounts of HIV-1 and HSV-2 and in HIV-1 and HSV-2 co-infected cervicovaginal lavage pools. The level of viral nucleic acids remained stable at 4 degrees C for 24h but decreased significantly when cervicovaginal lavages were stored at 20 degrees C and 30 degrees C, demonstrating that, cervicovaginal lavages to be quantified for viral RNA or DNA require, at minimum, immediate storage at 4 degrees C.

Cervix Uteri↗

PubMed1997

A histopathological study on localization of hepatitis C virus RNA in liver of chronic hepatitis C.

By in situ hybridization (ISH) method using digoxygenin (DIG)-labeled RNA probe, we proved the localization and replication of hepatitis C virus (HCV) RNA not only in the hepatocytes but also in the lymphocytes of the 52 liver tissues of chronic hepatitis C. We classified the staining patterns into four groups. The pattern of signal distribution significantly correlated with serum alanine aminotransferase (ALT) level, quantity of serum HCV RNA just before liver biopsy, histological grade of inflammatory activity, and stage of fibrosis by Spearman rank correlation. Moreover, we investigated the relation between the signals in the hepatocytes or lymphocytes and the effectiveness of interferon (IFN) therapy. Therefore, we suggest that ISH is a useful tool to evaluate the activity of chronic hepatitis C associated with serological and histopathological changes and is an important marker to assess the effectiveness of IFN treatment.

Adult↗

PubMed1992

Expression of Alu and 7SL RNA in Alzheimer's and control brains.

We investigated whether changes in expression of RNA polymerase III (pol III) or heterodisperse RNA polymerase II (pol II) transcripts hybridizing to Alu could be detected in Alzheimer's disease (AD). RNA samples obtained from AD and control brain tissues were examined by Northern analysis for Alu and 7SL RNA expression. All RNA samples contained a prominent band of approximately 300 nucleotides which corresponds to 7SL RNA, the Alu-homologous RNA component of the signal recognition particle. In addition, three small (i.e. less than 300 nucleotide) 7SL/Alu-hybridizing transcripts were detected. The two larger of the low molecular weight transcripts hybridized preferentially to the 7SL RNA probe, while the smallest transcript hybridized to the Alu probe. These transcripts and the heterodisperse RNA were variable in quantity and displayed a lack of correlation with AD.

Alzheimer Disease↗

PubMed2005

Isolation of Brucella abortus total RNA from B. abortus-infected murine RAW macrophages.

Brucella is a Gram-negative facultative bacterium that persists intracellularly in macrophages. However, the intracellular survival mechanisms used by Brucella are not fully understood. Isolation of Brucella RNA from infected macrophages has been challenging, and the inability to isolate sufficient Brucella RNA from infected macrophages has contributed to the failure in understanding bacterial transcriptional events. We describe the isolation of sufficient Brucella abortus RNA from its infective host cell environment using osmotic lysis and RNase and DNase digestion. This method takes advantage of the B. abortus cell envelope that protects bacterial RNA and DNA. The cell envelope of B. abortus was digested using SDS/proteinase K (PK) that, importantly, inhibits any residual RNase after digesting macrophage RNA permitting the extraction of B. abortus RNA. In our experiments, 4.5 microg of RNA was routinely isolated from 1 ml bacterial culture and 2-9 microg of bacterial RNA from infected macrophages without detectable host cell RNA or DNA contamination. The method is rapid and uses inexpensive, commonly available reagents. Total bacterial RNA was isolated in quantities sufficient for RT-PCR and microarray analysis.

Animals↗

PubMed1995

Detection of major bcr-abl gene expression at a very low level in blood cells of some healthy individuals.

The major bcr-abl fusion gene is presently seen as the hallmark of chronic myeloid leukemia (CML) and presumably as the cause of its development. Accordingly, long-term disappearance of bcr-abl after intensive therapy is considered to be a probable cure of CML. The nested reverse transcriptase-polymerase chain reaction (RT-PCR) provides a powerful tool for minimal residual CML detection. The RT-PCR was optimized by (1) increasing the amount of total RNA involved in the reverse transcription reaction to correspond to total RNA extracted from 10(8) cells, (2) using a specific abl primer in this reverse reaction, and (3) reamplifying 10% of the RT-PCR product in nested amplification. This optimized RT-PCR permitted us to detect up to 1 copy of RNA bcr-abl synthesised in vitro, mixed with yeast RNA in an equivalent quantity to 10(8) white blood cells (WBCs). Using this highly sensitive RT-PCR during the follow-up of CML patients, a signal was unexpectedly found in healthy controls. Therefore, a systematic study of the possible expression of bcr-abl RNA in the WBCs of healthy adults and children and in umbilical cord blood was undertaken. It showed the presence of bcr-abl transcript in the blood of 22 of 73 healthy adults and in the blood of 1 of 22 children but not in 22 samples of umbilical cord blood.

Adolescent↗

PubMed1981

Monocistronic transcription is the physiological mechanism of sea urchin embryonic histone gene expression.

We have examined histone gene expression during the early stages of sea urchin embryogenesis. The five histone genes expressed at that time are contained in tandem repetitive segments. It has been suggested that adjacent coding regions and their intervening spacer sequences are transcribed into large polycistronic messenger ribonucleic acid (RNA) precursors. We have subcloned into pBR322 deoxyribonucleic acid (DNA) sequences mapping either in the coding region, the 5' spacer, or the 3' spacer of the H2B histone gene. These clones were used to produce radioiodinated hybridization probes. We measured the steady-state quantity of H2B messenger RNA as well as spacer-specific RNA in the total RNA from embryos taken at various stages of development from fertilization to hatching of blastulae (0 to 22 h post-fertilization). Small amounts of RNA hybridizing to both spacer probes could be found. However, we show that these RNAs form mismatched hybrids with the spacer DNA and therefore cannot originate from the spacers present in the histone genes. We conclude that there is no detectable transcription of the spacer regions on either side of the H2B histone gene. The detection limit for RNA complementary to the 5' spacer sequence corresponds to a maximum of about three RNA molecules per cell, an amount shown to be far less than the projected steady-state pool size of a putative polycistronic transcript, if such a precursor were to be the obligatory transcript of the histone genes. (This conclusion was derived by using the known rates of production of H2B mRNA throughout early development [R. E. Maxson and F. H. Wilt, Dev. Biol., in press].) The physiologically relevant transcript of the histone genes in early development is therefore monocistronic and probably identical to the messenger RNA itself.

Animals↗

PubMed2003

FGL2 prothrombinase messenger RNA expression in gravid and nongravid human myometrium.

OBJECTIVE: Thrombin is a potent uterotonic agent that may play an important role in human parturition. FGL2 is a prothrombinase that cleaves prothrombin to thrombin. The purpose of this study was to determine FGL2 messenger RNA expression in human myometrium and to assess its relationship to the expression of EP3-6 prostaglandin receptor and inducible nitric oxide synthase. STUDY DESIGN: Myometrium was obtained from gravid women who underwent cesarean delivery (n = 19), from premenopausal (n = 10) and menopausal (n = 10) women who underwent hysterectomy, and from nonpregnant women who received 200 mg of misoprostol vaginally 12 hours before hysterectomy (n = 8). FGL2 messenger RNA expression was determined with the use of semiquantitative polymerase chain reaction and was compared with previously determined messenger RNA expression levels for EP3-6 and inducible nitric oxide synthase for the same samples. Statistical analysis was performed with the use of independent t test, Fisher exact test, analysis of variance, and linear regression, where appropriate. RESULTS: All samples that were analyzed contained demonstrable quantities of FGL2 messenger RNA. The highest FGL2 expression was found in gravid women, followed by women who were treated with misoprostol, by premenopausal nonpregnant women, and by menopausal women (P <.0001, analysis of variance). Significantly higher levels of FGL2 expression were found in the gravid women compared with the women who were treated with misoprostol (P <.001), the premenopausal nonpregnant women (P <.001), and the menopausal women (P <.0001). FGL2 messenger RNA expression correlated with EP3-6 and inducible nitric oxide synthase expression in all samples combined (r = 0.55, P <.0001; and r = 0.67, P <.001, respectively). CONCLUSION: The expression of FGL2 messenger RNA is increased in gravid myometrium compared with nonpregnant samples. Its expression significantly correlates to EP3-6 and inducible nitric oxide synthase expression. FGL2 prothrombinase expression may mediate the role of thrombin in human parturition.

Adult↗

PubMed1989

A continuous analog for RNA folding.

A linear segment in which a number of pairs of intervals of equal length are identified as potential stems is the subject of a folding problem analogous to inference of RNA secondary structure. A quantity of free energy (or equivalently, energy per unit length) is associated with each stem, and the various types of loops are assigned energy costs as a function of their lengths. Inference of stable structures can then be carried out in the same way as in RNA folding. More important, perturbation of stem lengths and energy densities (modelling various mutational processes affecting nucleotide sequences) allows the delineation of domains of stability of various foldings, through the explicit calculation of their boundaries, in a low-dimensional parameter space.

Mathematics↗

PubMed2006

Equal amounts of intracellular and virion-enclosed hepatitis C virus RNA are associated with peripheral-blood mononuclear cells in vivo.

BACKGROUND: Hepatitis C virus (HCV) replicating in peripheral-blood mononuclear cells (PBMCs) may represent an extrahepatic viral reservoir. Quantitation of HCV RNA with regard to its subcellular distribution and longitudinal course is needed for better understanding of the largely unexplored in vivo dynamics and potential pathogenetic significance of HCV in PBMCs. METHODS: Plasma and PBMCs from 30 patients coinfected with HCV and human immunodeficiency virus were evaluated in cross-sectional and longitudinal analyses, for up to 40 months. Differential extraction of virion-enclosed HCV RNA associated with cells was performed in parallel with extraction of total cellular HCV RNA. HCV RNA of either orientation was quantified by real-time polymerase chain reaction. RESULTS: HCV RNA was detected only in PBMCs from patients with viremia and at relatively stable quantities over time. Intracellular HCV RNA corresponding to ~60% of total cellular HCV RNA was strongly correlated with virion-enclosed HCV RNA but was only weakly associated with viral loads in plasma. In contrast, the ratio of HCV RNA load in PBMCs versus that in plasma was patient specific and stable over time. CONCLUSIONS: The substantial and patient-specific amounts of intracellular HCV RNA found by the present study support a concept of low-level replication in PBMCs. There was no evidence for persistent HCV infection in PBMCs after clearance of viremia in plasma.

Adult↗

PubMed1995

Relation between serum hepatitis C virus-RNA levels and efficacy of interferon-beta therapy.

We performed two courses of interferon-beta (IFN-beta) to a child with chronic hepatitis C. A complete response was not obtained by the first interferon treatment, however, the results of the second treatment differed from those of the first. Hepatitis C virus (HCV)-RNA remained negative and both aspartate aminotransferase and alanine aminotransferase levels remained normal after completion of the second course. From these results we estimated that HCV-RNA levels before IFN therapy could be significantly associated with the efficacy of this treatment. The serum level of HCV-RNA was 10(6) copies/50 microL before the first treatment, but was 10(3) copies/50 microL before the second course. We conclude that IFN therapy to children with hepatitis C should always be directed at providing a cure. Even if the clinical effects of the first course are minimal decreasing quantities of HCV-RNA still offer hope for cure by subsequent readministration.

Adolescent↗

PubMed1979

[The molecular biology of aging -- 15: Structure and function of the genetic apparatus in aging. II. Synthesis and metabolism of RNA].

With aging in different organs there are similar or tissue specific changes of the ribonucleic acids in respect of the quantity, the spectrum of different RNA species and the metabolism of RNA. Among these processes the template function of the chromatin and the behaviour of the RNA polymerases are important influencing factors. In aging the sensitivity of the RNA synthesis against hormonal stimulation is decreased in the most cases. A survey about the results of many in vivo and in vitro experiments of the literature is given. The opinion is advocated that the problems of the gene regulation and gene expression are a important field of work in the experimental gerontology.

Adrenal Cortex Hormones↗

PubMed1979

Isolation by differential and zonal centrifugation of minicells segregated by Escherichia coli.

Minicells segregated from Escherichia coli chi925 carrying a drug-resistance plasmid were separated from nucleated cells by differential centrifugation and purified by rate-zonal centrifugation in sucrose gradients. Minicells purified in this way were capable of donating the plasmid to nucleated cells. They also incorporated thymidine, uridine and methionine into macromolecules. Methods are described for purification of plasmid-containing minicells on a scale large enough to allow isolation of DNA, DNA polymerase and RNA polymerase in sufficient quantities for studies of enzymes involved in replication and transcription of plasmid DNA.

Cell Fractionation↗

PubMed1994

Cloning of a pig glucose transporter 4 cDNA fragment: use in developing a sensitive ribonuclease protection assay for quantifying low-abundance glucose transporter 4 mRNA in porcine adipose tissue.

A 246-bp fragment of porcine glucose transporter 4 (GLUT4) cDNA was cloned by polymerase chain reaction (PCR) from porcine adipose tissue RNA. Nucleotide sequences 1-138 and 139-246 of the GLUT4 cDNA share 78% sequence identity with exon 4a and 91% sequence identity with exon 4b of the human GLUT4 gene, respectively. The GLUT4 cDNA fragment was subcloned into pGEM-4Z vector to synthesize a highly specific riboprobe that hybridized only to human GLUT4 cDNA but not to human glucose transporter 1 (GLUT1) cDNA. Northern blot analysis of total RNA revealed the presence of a single transcript of 2.8 kb in porcine adipose tissue. Cloning a fragment of the GLUT4 cDNA enabled us to develop a ribonuclease protection assay for detecting porcine GLUT4 mRNA. The ribonuclease (RNase) protection assay is highly reproducible and retains a sensitivity level to as little as 2 pg of GLUT4 mRNA. The standard curve was linear between 2 and 128 pg of sense-strand GLUT4 RNA (r = .994). The ability to detect small quantities of GLUT4 mRNA is important when the abundance of GLUT4 mRNA is low and the quantity of tissue is limiting (e.g., when RNA is extracted from cultured adipose tissue). When porcine adipose tissue explants were cultured in the presence of insulin (10 ng/mL), GLUT4 mRNA abundance was increased. Development of a sensitive assay to quantify GLUT4 mRNA in porcine adipose tissue will enable us to conduct studies to increase our understanding of the molecular mechanisms by which porcine somatotropin (pST) regulates GLUT4 gene expression.

Adipose Tissue↗

PubMed1980

Cloning of avian tumor virus DNA fragments in plasmid pBR322: evidence for efficient transcription in E. coli from a virus-coded promoter.

Two avian tumor virus DNA fragments of 4.2 and 3.2 kb were inserted into pBR322 in the two possible orientations for each fragment. In the Escherichia coli host cells, RNA polymerase initiates transcription of large quantities (up to 0.5 to 1% of total E. coli RNA) of virus-specific RNA in the recombinant plasmids carrying the 4.2-kb fragment (pATV-6) but not in pATV-2 which contains the 3.2-kb fragment. Two SacI cleavage sites flank the putative promoter in the 4.2-kb viral insert. Deletion in the 1.2-kb SacI fragment obliterated the ability of pATV-6 to synthesize viral RNA. Digestion of the 1.2-kb SacI fragment with PvuI generates two fragments of 0.63 and 0.57 kb. Deletion of the 0.57-kb but not the 0.63-kb PvuI-SacI fragment completely eliminated the ability of the recombinant to synthesize viral RNA. These results strongly suggest that viral RNA in E. coli transcription is indeed initiated at a size present in the viral genome and that this site is localized in the 0.57-kb PvuI-SacI fragment.

Cloning, Molecular↗

PubMed1991

Detection of phocid distemper virus RNA in seal tissues using slot hybridization and the polymerase chain reaction amplification assay: genetic evidence that the virus is distinct from canine distemper virus.

Slot hybridization and the polymerase chain reaction (PCR) after reverse transcription (RT) were used to detect RNA extracted from tissues of seals after naturally occurring disease and experimental infection with phocid distemper virus (PDV). A phosphoprotein (P) gene-specific cDNA served as a probe for both slot hybridization and the identification of PCR-generated fragments by Southern blotting. As primers for the PCR assay PDV P gene-derived oligonucleotides were used. Hybridization, PCR and partial nucleic acid sequence analysis clearly demonstrated that PDV is a distinct virus (most closely related to canine distemper virus) within the morbillivirus group. PCR, when combined with Southern blot hybridization, was clearly superior to slot hybridization and more sensitive than cell culture isolation and immunofluorescence assays for the detection of virus in tissues. Considerable amounts of viral RNA could be demonstrated in the lungs and spleens. In experimentally infected animals a large quantity of virus-specific RNA was additionally found in colon samples. Using RT-PCR in combination with Southern blotting. PDV could be demonstrated in buffy coat cells using a simple and fast cell lysis procedure.

Animals↗