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Salt dissociation of nuclear particles containing DNA-like RNA. Distribution of phosphorylated and nonphosphorylated species.

Electrophoresis of proteins from nuclear particles containing DNA-like RNA gave a pattern with 45 bands. The possibility that some of these proteins arose by contamination with ribosomes, chromatin, or soluble nuclear proteins was examined and eliminated. The fate of the proteins of the particles was studied after partial dissociation with 0.25 and 0.70 M NaCl. The individual proteins were released progressively and in different quantities. A group of easily released species (75 and 95% removed with 0.25 and 0.70 M NaCl) was demonstrated. This group contained 8 species between 29,000 and 39,000 daltons which represented approximately one-half of the total number of molecules. It is suggested that they are bound to repetitive sequences of the RNA. At least 30 and 60% of the other proteins were released at 0.25 and 0.70 M NaCl, respectively. There were no specific proteins tightly bound to the RNA, unless the nature of the remaining species is different from that of the released ones of the same molecular weight. The phosphorylated proteins were more tightly bound to the RNA than the nonphosphorylated species of similar molecular weight. In several instances, the 32-P radioactivity was associated with quantitatively minor bands of proteins.

Animals↗

Nucleolar morphological rearrangement related to transcriptional and replicative state in Burkitt lymphoma cells.

The specific silver staining of nucleolar region organizers was applied to Daudi lymphoma control and interferon alpha-treated cells. Isolated nuclei from control and treated samples were used for the kinetic analysis of in vitro RNA and DNA synthesis. Results have shown that interferon treatment induces a reduction of the transcriptional and replicative activities within 90 min. Concomitant to these results is the modification of the organization of nucleoli. Intensity and distribution of silver grains are, in fact, different in treated cells nucleoli as compared to those of controls. Thus, the number and the arrangement of granules could be related to the functional state of the cells suggesting that the transient cascade of interferon-generated signals involves also modulation of nucleolar structure and function in accordance with the hypothesis of a relationship of cell proliferation rate to silver-stained nucleolar protein quantity.

Amanitins↗

Quantitative measurement of dihydrouridine in RNA using isotope dilution liquid chromatography-mass spectrometry (LC/MS).

A method has been developed for the microscale determination of 5,6-dihydrouridine, the most common post-transcriptional modification in bacterial and eukaryotic tRNA. The method is based on stable isotope dilution liquid chromatography-mass spectrometry (LC/MS) using [1,3-15N2]dihydrouridine and [1,3-15N2]uridine as internal standards. RNA samples were enzymatically digested to nucleosides before addition of the internal standards and subsequently analyzed by LC/MS with selected ion monitoring of protonated molecular ions of the labeled and unlabeled nucleosides. Sample quantities of approximately 1 pmol tRNA and 5 pmol 23S rRNA were analyzed for mole% dihydrouridine. Dihydrouridine content of Escherichia coli tRNASer(VGA) and tRNAThr(GGU) as controls were measured as 2.03 and 2.84 residues/tRNA molecule, representing accuracies of 98 and 95%. Overall precision values for the analyses of E. coli tRNASer(VGA) and E. coli tRNAThr(GGU), unfractionated tRNA from E. coli and 23S rRNA from E. coli were within the range 0.43-2.4%. The mole% dihydrouridine in unfractionated tRNA and 23S rRNA from E. coli were determined as 1.79 and 0.0396%, corresponding to 1.4 and 1.1 residues/RNA molecule respectively.

Chromatography, High Pressure Liquid↗

Sequence analysis of RNA species synthesized by Q beta replicase without template.

Q beta replicase amplifies certain short-chained RNA templates autocatalytically with high efficiency. In the absence of extraneously added template, synthesis of new RNA species by Q beta replicase is observed under conditions of high enzyme and substrate concentrations and after long lag times. Even under identical conditions, different RNA species are produced in different experiments. The sequences of several independent template-free products have been determined by cloning their cDNAs into plasmids by a novel cloning procedure. Their nucleotide chain lengths are small, ranging from 25 to about 50 nucleotides. While their primary sequences are unrelated except for the invariant 5'-terminal G and 3'-terminal C clusters, their tentative secondary structures show a common principle: both their plus and minus strands have a stem at the 5' terminus, while the 3' terminus is unpaired. Direct accumulation of sufficient quantities of early template-free synthesis products by Q beta replicase is prevented by the inherent irreproducibility of the synthesis process and by the rapid change of the products during amplification by evolution processes, but large amounts of such RNA can be synthesized in vitro by transcription from the cDNA clones. RNA species produced in template-free reactions replicate much more slowly than the optimized RNA species characterized previously. These experimental results illustrate how biological information can be gained in small bits by trial and error.

Base Sequence↗

Biochemical studies of isolated hamster tracheal epithelium.

The epithelial lining of respiratory air passageways is a primary target tissue for toxicity and carcinogenesis in man and in animal models of human disease. The importance of this target tissue was the basis for development of methods to study its biochemistry, and with this information to distinguish the unique properties of this tissue from properties common to all cell types. Biochemical methods employed labeling of macromolecules in isolated hamster treacheas during brief (less than 4 hr) incubation in vitro. Studies of RNA metabolism in isolated tracheas demonstrated a pattern of maturation of ribosomal RNA like that shown for other cell types. Alterations in RNA metabolism were observed in isolated tracheas obtained from vitamin A-deficient hamsters and hamsters previously treated by intratracheal administration of benzo[a]pyrene (BP) plus ferric oxide (Fe2O3) in vivo. Studies with toyocamycin, actinomycin D, and alpha-amanitin, all inhibitors of RNA metabolism, were performed to characterize the class of RNA molecules with a decreased proportion of labeling in tracheas from vitamin A deficient hamsters. In another series of experiments, BP was shown to bind to DNA in epithelial cells of isolated tracheas. The quantity of BP binding was increased by prior intratracheal treatment of hamsters with BP plus Fe2O3 in vivo, this induced binding was inhibited by addition of 7,8-benzoflavone to the incubation medium. Increased BP binding was also observed in isolated tracheas from hamsters believed to be in states of increased susceptibility to respiratory carcinogenesis in vivo. The results show that biochemical studies are feasible with this tissue. Furthermore, a number of questions of importance with regard to this target epithelium are best studied directly in its constituent cells.

Adenosine↗

Capacity of ringed sideroblasts to synthesize nucleic acids and protein in patients with primary acquired sideroblastic anaemia.

Bone marrow cells from two patients with primary acquired sideroblastic anaemia were studied using the technique of electron microscope autoradiography. Whereas a significant proportion of erythroblasts containing small to moderate quantities of intramitochondrial deposits incorporated [3H]thymidine into DNA, the majority of cells containing large quantities of intramitochondrial deposits did not. These results are consistent with the idea that several of the early polychromatic cells with large quantities of deposits become arrested in their progress through the cell cycle. The data also revealed that the accumulation of increasing quantities of iron-containing material within the mitochondria of an erythroblast was frequently (but not invariably) associated with and was possibly responsible for (I) a depression of RNA synthesis and (2) a more marked depression of protein synthesis. A small proportion of the erythroblasts showing a depression of protein synthesis also displayed various non-specific ultrastructural abnormalities indicative of cellular injury such as the presence of intranuclear clefts, nuclear membrane-associated myelin figures and lipid-laden intracytoplasmic vacuoles.

Aged↗

The morphological and functional differentiation of the alimentary canal of the pig during ontogeny. I. Development and differentiation of the fundic portion of the stomach.

Several periods of intensive growth are observed in the development of the fundus of pig's stomach: at the end of the 2nd and the beginning of the 3rd month of embryogenesis, the periods shortly prior to and after birth, and between the 10th and 20th day following birth. The gastric pits are formed at about the 45th day of embriogenesis. At the beginning of the third month the fundic glands are formed. The parietal cells first differentiate and are found in 60-day old embryos. The mucous neck cells are differentiated at the beginning of the 3rd month but secretion of mucoid substance starts on about the 45th to 50th day of the prenatal development. With increasing age of the pigs the quantity of PAS-positive substances in the surface epithelium, as well as in the mucous neck cells gradually increases. The differentiation of the chief cells rich in RNA occurs approximately on the 90th day of the prenatal development.

Age Factors↗

Conjugal deoxyribonucleic acid replication by Excherichia coli K-12: effect of chloramphenicol and rifampin.

Conjugal replication of R64-11 deoxyribonucleic acid (DNA) and the concomitant transfer of R64-11 DNA to DNA-deficient minicells are dependent upon processes that are inhibited by rifampin and chloramphenicol. The rifampin-sensitive product is not present in vegetatively growing cells and is needed to initiate both conjugal DNA replication in donor cells and DNA transfer to recipient minicells. If the rifampin-sensitive product is a ribonucleic acid (RNA) molecule (rather than RNA polymerase itself), our data indicate that this RNA species required for initiation of conjugal activity does not need to be translated into a protein product. The chloramphenicol-sensitive product(s) is present in vegetatively growing cells in sufficient quantity to permit most donor cells to carry out one round of plasmid conjugal replication and transfer. The initiation of second and subsequent rounds of conjugal replication and transfer are dependent on the synthesis of both the rifampin-sensitive and chloramphenicol-sensitive products. Our results demonstrate a correspondence between the amount of conjugal DNA replication in the donor and the amount of DNA transferred to recipient minicells under all conditions, and therefore suggest but do not prove that plasmid transfer is dependent on conjugal DNA replication. The results also add additional proof that R64-11 transfer to minicells is discontinuous. All of these results are discussed in regard to further refinements of old models for the mechanism of conjugal transfer as well as a more radical departure from current dogma.

Bacterial Proteins↗

A molecular survey of hypoxanthine-guanine phosphoribosyltransferase deficiency in man.

We characterized 24 unrelated patients with a deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) in an attempt to better understand the nature and spectrum of mutations that underlie this prototype-inherited disease. Lymphoblast cell lines derived from each patient were analyzed at multiple molecular levels including the structure and function of the residual HPRT enzyme, messenger RNA (mRNA), and gene. Our studies demonstrate the following: (a) at least 16 of the 24 patients represent unique and independent mutations at the HPRT structural gene; (b) the majority of cell lines have normal quantities of mRNA but undetectable quantities of enzyme; (c) 33% of patients retain significant quantities of structurally altered, functionally abnormal, HPRT enzyme variants; and (d) a minority of patients are void of both enzyme and mRNA, possibly representing examples of aberrations in gene expression. Our studies provide direct evidence for marked genetic heterogeneity in this disorder and illustrate the kinds of mutations and mutational consequences that underlie inherited disease in humans.

Cell Line↗

Viral load quantitation of SARS-coronavirus RNA using a one-step real-time RT-PCR.

INTRODUCTION: Severe acute respiratory syndrome (SARS) is an emerging infectious disease that first occurred in humans in the People's Republic of China in November 2002 and has subsequently spread worldwide. A novel virus belonging to the Coronaviridae family has been identified as the cause of this pulmonary disease. The severity of the disease combined with its rapid spread requires the development of fast and sensitive diagnostic assays. RESULTS: A real-time quantitative RT-PCR was designed in the nsp11 region of the replicase 1B domain of the SARS-coronavirus (SARS-CoV) genome. To evaluate this quantitative RT-PCR, cRNA standards were constructed by in vitro transcription of SARS-CoV Frankfurt 1 RNA using T7 RNA polymerase, followed by real-time RT-PCR. The assay allowed quantitation over a range of 10(2) to 10(8) RNA copies per reaction. CONCLUSIONS: Extrapolated to clinical samples, this novel assay has a detection range of 10(4) to 10(10) copies of viral genome equivalents per millilitre. In comparison to the current de facto cRNA Artus Biotech standard, the in-house cRNA standard gives a 100-fold higher absolute quantity, suggesting a possible underestimation of the viral load when using the Artus Biotech standard.

Animals↗

Analysis of immunoglobulin genes: DNA/RNA hybridization with immunoglobulin kappa-chain mRNA and isolation and translation of hybridized RNA.

Immunoglobulin kappa-chain mRNA was hybridized with DNA in order to assess the kappa-gene frequency. Kappa-mRNA was purified from membrane-bound ribosomes of mouse myeloma MOPC-41 by poly (U) chromatography and isolation of a 13S RNA by successive sucrose density gradient centrifugations. The RNA coded for kappa-chain precursor molecules in cell-free protein synthesis and essentially no other proteins. MOPC-41 kappa-mRNA hybridized with MOPC-41, MPC-11, and Krebs DNA with the same kinetics: the majority of the hybrids was formed with rare or unique DNA sequences (Cot/2 450 to 900), a small portion with highly repetitive sequences (Cot/2 5--6). The slow hybrids were well matched and the rapid hybrids were mismatched by about 4%, regardless of the DNA used. It was further investigated whether the rapid hybrids contained translatable kappa-mRNA or were due to impurities in the RNA preparations. Kappa-mRNA and globin-mRNA (as an internal standard for a unique transcript) were hybridized with DNA to Cot 20 or 48, the hybridized and unhybridized RNA were isolated by hydroxyopatite-urea chromatography and, after removal of the DNA, translated in a cell-free system. The cell-free products were analyzed by SDS-polyacrylamide gel electrophoresis and immunoprecipitation. It was found that approximately equal quantities of translatable kappa- and globin-mRNA were hybridized maximally 1.7%). The results do not support the hypothesis that kappa-mRNA is a transcript of both repetitive and unique DNA sequences.

Animals↗

Encapsidation of genetically engineered poliovirus minireplicons which express human immunodeficiency virus type 1 Gag and Pol proteins upon infection.

The use of recombinant viruses for the expression of a wide array of foreign proteins has become commonplace during the last few years. Recently, we have described the construction and characterization of chimeric human immunodeficiency virus type 1 (HIV-1)-poliovirus genomes in which the gag and pol genes of HIV-1 have been substituted for the VP2 and VP3 capsid genes of the P1 capsid precursor region of poliovirus. Transfection of these RNAs into tissue culture cells results in replication of the RNA genome and expression of HIV-1-P1 fusion proteins (W. S. Choi, R. Pal-Ghosh, and C. D. Morrow, J. Virol. 65:2875-2883, 1991). Here we report on the encapsidation and amplification of the minireplicons to obtain sufficient quantities for biological characterization. To do this, HIV-1-poliovirus minireplicon genomes containing the gag or pol gene were transfected into cells previously infected with a recombinant vaccinia virus (VV-P1) which expresses the poliovirus capsid precursor protein, P1 (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 65:2088-2092, 1991). The chimeric minireplicons replicated and expressed the appropriate HIV-1-P1 fusion proteins as determined by immunoprecipitation with HIV-1-specific antibodies. The encapsidated genomes were isolated by ultracentrifugation. Reinfection of cells with the encapsidated chimeric RNA genomes resulted in expression of the HIV-1-Gag-P1 or HIV-1-Pol-P1 fusion protein. Serial passaging of the encapsidated chimeric HIV-1-poliovirus genomes was accomplished by coinfecting cells with the encapsidated minireplicons and VV-P1, resulting in stocks of the encapsidated minireplicons. Northern (RNA) blot analysis of passaged material revealed that no detectable deletions of the chimeric genomes occurred during 14 serial passages. Infection of cells by the encapsidated minireplicons was blocked by antipoliovirus antibodies. Coinfection of cells with encapsidated minireplicons and type 1 Sabin poliovirus resulted in encapsidation of the chimeric genomes by wild-type poliovirus as measured by immunoprecipitation of the HIV-1-P1 fusion proteins with HIV-1-specific antibodies. The results of this study demonstrate the encapsidation of poliovirus minireplicons which express foreign proteins and point to the future use of this system as a potential vaccine vector.

Capsid↗

Oral and systemic factors associated with increased levels of human immunodeficiency virus type 1 RNA in saliva.

OBJECTIVE: The purpose of this investigation was to quantify human immunodeficiency virus type-1 (HIV-1) RNA in saliva and plasma and identify factors associated with increased salivary viral load. STUDY DESIGN: Forty HIV-1-seropositive adults underwent oral examinations to assess mucosal and periodontal health. Whole saliva was evaluated for HIV-1 RNA titer and occult blood. Plasma viral load, CD4 cell count, HIV-1 staging, and antiretroviral therapy data were obtained from medical records. Associations between salivary titers and oral/systemic parameters were analyzed by means of t tests, Wilcoxon signed rank tests, Pearson's correlation coefficient, and analysis of covariance. RESULTS: Forty-two percent of the subjects had detectable salivary HIV-1 RNA. Oral titers were highly correlated with plasma viral levels (r = 0.51, P <.01). HIV-associated periodontal disease (in particular, linear gingival erythema), severe gingival inflammation, and absence of antiretroviral therapy were associated with high salivary titers (P <.01). CONCLUSIONS: Substantial quantities of HIV-1 can be shed in the oral cavity, particularly when inflammatory conditions are present. Salivary titer may be a useful indicator of systemic viral burden.

Adult↗

Cell-free translation of American hop latent virus RNA.

The RNA of American hop latent virus (AHLV) has a molecular size of 7.7 kb measured in agarose gels. Translation products of AHLV RNA in rabbit reticulocyte and wheat-germ cell-free systems ranged in size up to 200 kD. Time-course experiments indicated that a 36 kD peptide, immunoprecipitated by antivirion sera, was synthesized early followed by the sequential appearance of peptides of increasing Mr. An excess of the amino-acid analogue L-canavanine for arginine specifically reduced the quantity of the 36 kD peptide and induced synthesis of a 38-kD peptide in both rabbit reticulocyte and wheat germ. Translation products were not altered by the addition of a reducing agent, and no product appeared to be produced due to limitation of tRNA species or by readthrough. Synthesis of AHLV RNA-directed peptides was blocked by the cap analogue m7G5'ppp5'G, suggesting the presence of a cap structure at the 5' terminus.

Animals↗

Control of TGEV mRNA transcription.

Coronavirus proteins are translated from a nested set of subgenomic mRNAs which have common 3' termini with unique 5' extensions. Evidence suggests that coronavirus mRNAs are generated by a mechanism of leader primed transcription. Leader RNA binds to consensus sequences upstream of each gene on full length negative strand viral RNA and transcription proceeds to the 5' end of the negative strand to produce the nested set of mRNAs. Even though this gives rise to polycistronic mRNA species only the 5' extension of each mRNA is translated to give the viral proteins. The leader RNA for TGEV is about 90 nucleotides long and contains the sequence which recognises the leader binding sites on the negative strand RNA. Evidence suggests that the length of the leader binding sequence may be involved in transcriptional control of individual mRNAs. In order to investigate this a virus specific mRNA isolation method was developed to measure the relative amounts of mRNAs synthesised during an infection of LLC-PK1 cells with TGEV (strain FS772/70). Thus the relative quantity of each mRNA can be determined and correlated with the variation in size of the leader binding site.

Animals↗

A new type of virus from cultured Drosophila cells: characterization and use in studies of the heat-shock response.

An infectious virus isolated from the cultured Drosophila melanogster cell line, Schneider 2-L, appears to be a member of a new group of animal viruses. The virus, HPS-1 has a genome composed of a single segment of double-stranded RNA approximately 6 kb in length. Virions are particles approximately 36 nm in diameter. They contain only two proteins and no lipid coat. The major protein, presumably the viral coat protein, is 120,000 daltons. A 200,000 dalton protein is present in much lower quantities. Two other proteins, synthesized in virus-producing cells, are encoded in the viral genome but not included in the mature virion. Synthesis of viral proteins is not affected by the heat-shock-induced translational control that inhibits translation of most normal mRNAs but allows protein synthesis on heat-shock mRNAs. The viral mRNAs thus appear to share the structural features of heat-shock mRNAs which permit heir translation in heat-shocked cells. Viral RNA serves as a probe to study heat-shock translational control. Cells heat-shocked in the presence of actinomycin D cannot transcribe heat-shock mRNA and therefore cannot make heat-shock proteins. Although these cells are making neither heat-shock mRNA nor heat-shock protein, the translational control appears fully induced. The normal cell proteins are not made in these cells but viral proteins are synthesized. These results indicate that the heat shock-induced proteins are not components of the translational control mechanism.

Animals↗

Rapid diagnostic method for detection of mumps virus genome by loop-mediated isothermal amplification.

Most mumps patients are clinically diagnosed without any virological examinations, but some diagnosed cases of mumps may be caused by other pathogens or secondary vaccine failure (SVF). To clarify these issues, a sensitive, specific, and rapid diagnostic method is required. We obtained 60 salivary swabs from 34 patients with natural infection during the course of the illness, 10 samples from patients with vaccine-associated parotitis, and 5 samples from patients with SVF. Total RNA was extracted and subjected to reverse transcription-PCR (RT-PCR) and loop-mediated isothermal amplification (LAMP) for genome amplification. We detected mumps virus RNA corresponding to 0.1 PFU by LAMP within 60 min after RNA extraction, with the same sensitivity as RT-nested PCR. Mumps virus was isolated in 30 of 33 samples within day 2, and mumps virus genome was amplified by LAMP in 32 of them. The quantity of virus titer was calculated by monitoring the time to reach the threshold of turbidity. The viral load decreased after day 3 and was lower in patients serologically diagnosed as having SVF with milder illness. Accuracy of LAMP for the detection of mumps virus genome was confirmed; furthermore, it is of benefit for calculating the viral load, which reflects disease pathogenesis.

Animals↗