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At least 145 records · Page 8Linked to original sources
PubMed1971

Ribosomes and Polysomes in Cucumber Leaves during Growth and Senescence.

The quantity of RNA in the ribosomal fraction of the first leaf of cucumber (Cucumis sativus) increases during growth, reaches a maximum before the final fresh weight is attained, and then decreases. The main changes are in the free ribosome fraction, the quantity of membrane-bound ribosomes remaining about constant. Few 65.5S chloroplast ribosomes are present in small leaves; however, they increase in quantity rapidly during growth and form about half of the ribosomes present in the mature fully green leaf. The cytoplasmic ribosomes have a sedimentation coefficient of 77.6S. Ribonuclease-sensitive polysomes were present in leaves of all ages except possibly the very oldest. The proportion of ribosomes in polysome form decreases during growth and then remains roughly constant during senescence. Following maturation of the leaf, the rate of incorporation of (32)P into ribosomal-fraction RNA begins to decline. This decline could account for the loss of ribosomes during the early stages of senescence. The possibility that leaf ribonuclease might be responsible for the final, more rapid loss of RNA, is discussed.

Journal Article↗
PubMed1974

Isolation and sequence determination of the 3'-terminal regions of isotopically labelled RNA molecules.

The method which was developed for the selective isolation of 3'-terminal polynucleotides from large RNA molecules on columns of cellulose derivatives containing covalently bound dihydroxyboryl groups has been modified and adapted for use on radioactively labelled RNAs. The 3'-terminal polynucleotide fragments which result from specific ribonuclease digestion of isotopically detectable quantities of RNA can be selectively obtained in both high yield and purity by the modified procedure and can be subsequently analyzed by standard electrophoretic and chromatographic techniques. In addition, when the extent of enzymatic fragmentation of the RNA is controlled, the procedure permits the selective isolation of discrete "sets" of fragments of variable chain length, all of which derive from the 3'-terminus of the RNA molecule. These overlapping polynucleotides can be used directly to obtain extensive sequence information regarding the primary structure in the 3'-region of the RNA.

Autoradiography↗
PubMed2005

Feasibility of RNA collection for micro-array gene expression analysis in the treatment of cervical carcinoma: a scientific correlate of RTOG C-0128.

PURPOSE: To determine the feasibility of RNA collection in a multi-institutional cooperative group setting to be utilized for micro-array gene expression analysis, and to describe the methodology. METHODS: RTOG C0128, a phase I-II, protocol was designed to look at the safety and efficacy of external beam radiation therapy to 45 Gy with concomitant 5-FU and cisplatin chemotherapy, brachytherapy to deliver 85 Gy to point A, and Celecoxib at 400 mg twice daily for 1 year. Patients had the option of participating in a tissue collection portion of the protocol to be utilized for micro-array gene expression analysis before treatment and at the time of the first implant. RNA quality was determined by two parameters: the absorbance ratio at 260 nm/280 nm, and by the ratio of the integrated peak of 28S RNA to 18S RNA after gel electrophoresis. RESULTS: From August 2001 to March 2004, 84 patients were accrued to the trial, and tissue was obtained prior to initiation of therapy on 34 patients (40%). FIGO stages for the patients who provided tissue were IB (23%), II (57%), and IIIA-IVA (20%). Additionally, biopsies were obtained at the time of the first implant from 22 of the accrued patients making paired samples available on 26% for RNA extraction and micro-array gene expression analysis. The mean +/- SEM amount of tissue obtained pretreatment was 97 +/- 13 mg compared with 51 +/- 8 mg for tissue obtained at the time of the first implant (P = 0.009). The mean total RNA extracted from the samples prior to treatment was 119 +/- 19 microg versus 35 +/- 6 microg at the time of the first procedure (P = 0.001). The RNA quality was assessed via the absorbance ratio at 260 nm divided by 280 nm. The mean values pretreatment and at first implant were 1.87 +/- 0.07 versus 1.66 +/- 0.11, respectively (P = 0.002); however, the integrated peak of 28S RNA to 18S RNA after gel electrophoresis was not significantly different (P = 0.26). CONCLUSIONS: RNA extraction for gene expression analysis can be successfully performed in the multi-institutional cooperative group setting. Fresh tissue samples were obtained on 40% of accrued patients prior to treatment. The amount of biopsy material and the quantity of RNA extracted were greater prior to treatment compared with the first implant. The quality of RNA was superior prior to treatment as measured by the ratio of absorbance at 260/280 nm. These results indicate that gene expression analysis is feasible in the cooperative group setting utilizing amplification techniques for the RNA. Hopefully, this will allow for improvement in prognosis, therapeutic development, and correlation with acute and late toxicities in patients with cancer.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1993

Rare renal transcription of the atrial natriuretic factor gene in rats. Demonstration by polymerase chain reaction.

Renal synthesis of a peptide homologous to atrial natriuretic factor (ANF) has been demonstrated. The aim of the present study was to determine if transcription of the ANF gene occurs in the kidney. Rat renal RNA was extracted from whole kidneys, and, separately, from the cortex and outer and inner medulla of rat kidneys. Probing with rat ANF-cDNA did not reveal a detectable message in Northern blot analysis, even when large quantities of RNA were used at low stringency hybridization conditions. Therefore, reverse transcription (RT) followed by 35 cycles of polymerase chain reaction (PCR) was used to search for the renal message for ANF. Two 21-mer primers encompassing the 450 base pairs (bp) of the coding region of the gene were used. Each cycle consisted of annealing at 56 degrees C, extension at 72 degrees C, and denaturation at 94 degrees C. The PCR product was proven to be identical to the ANF gene by high stringency hybridization, which revealed the expected 450-bp hybrid band. Furthermore, the sequence of this product was identical to that of the coding region of the ANF gene. We used an RNA-specific PCR to obtain this band as a single reaction product. We conclude that the transcript of the ANF gene exists in the kidney, at extremely low levels. The low abundance of the RNA message raises major concerns about its physiologic relevance. Direct evidence for the translation of this transcript, and its quantification and localization, is still required to determine its significance.

Animals↗
PubMed2000

Binding affinity of T7 RNA polymerase to its promoter in the supercoiled and linearized DNA templates.

A promoter competition assay was used to measure the stability of T7 RNA polymerase with its promoter. When T7 RNA polymerase was incubated with GTP for 5 minutes before the elongation of transcription in either the supercoiled or linearized template, the half-lives of T7 RNA polymerase-DNA complexes were reduced. The transcription product increased when T7 RNA polymerase preincubated with GTP in the supercoiled DNA but not in the linearized DNA template. On the other hand, preincubation of ATP with T7 RNA polymerase decreased the stability of T7 RNA polymerase with the supercoiled DNA, but did not affect the stability of T7 RNA polymerase with the linearized DNA. Furthermore, the production of RNA transcript was increased when T7 RNA polymerase was incubated with ATP in either supercoiled or linearized template before transcription elongation. This study is important to understand the relationship between the transcription initiation and the stability of the ternary complex, and to produce large quantities of RNA transcript in vitro.

Adenosine Triphosphate↗
PubMed1986

[Changes in the content of pepsinogen messenger RNA in the gastric mucosa of rats during N-methyl-N'-nitro-N-nitrosoguanidine-induced carcinogenesis].

Pepsinogen mRNA is shown to be the major fraction of rat poly (A)+RNA. It codes polypeptide with molecular weight of about 45 kD. Changes in the pepsinogen mRNA content at the early stage of carcinogenesis are nonspecific and are due to the toxic effect of MNNG. Steady shifts in the quantity of pepsinogen mRNA are found between the 1st and 3d months. Pepsinogen mRNA content decreases down to the half of the normal one between the 3d and 6th months. A quantity of RNA capable to be a template for pepsinogen synthesis is reduced by more than 90% in the MNNG induced tumour. The pepsinogen production defect in gastric mucosa neoplasia is mainly due to pepsinogen mRNA synthesis damage.

Animals↗
PubMed2001

Radioactive 33-P probes in hybridization to glass cDNA microarrays using neural tissues.

cDNA microarrays are becoming widespread tools in the study of complex gene expression patterns with applications using cells lines, animal model systems, and human disease. Glass cDNA microarrays using fluorescent labeled cDNA probes require a large amount of input RNA usually not available in many neuroscience applications. Here we demonstrate a technique for the use of 33-P labeled cDNA probes in hybridizations to the same glass cDNA arrays used for fluorescent applications. This approach allows the use of low quantities of RNA, common phosphoimaging scanners, data acquisition software, and standard DNA and RNA labeling protocols, while being consistent and interchangeable with glass-based cDNA array technology.

DNA Probes↗
PubMed1975

Preparation and preliminary characterization of purified ovalbumin messenger RNA from the hen oviduct.

Preparation of milligram amounts of purified ovalbumin mRNA was accomplished by a sequential combination of precise sizing techniques with the selective purification of the poly(A) containing RNA by either affinity chromatography or adsorption to nitrocellulose filters. Several new techniques were applied to the purification of ovalbumin mRNA including Sepharose 4B chromatography and agarose gel electrophoresis in the presence of 6 M urea at pH 3.5. All the procedures used were adapted on a preparative sacle to the fractionation of large quantities of RNA. The purity of the ovalbumin mRNA was assessed by several independent criteria. (1) Purified ovalbumin mRNA migrated as a single band during both agarose-urea and formamide-polyacrylamide gel electrophoresis at pH 3.5 and 7.4, respectively. A single absorbance peak containing all of the ovalbumin mRNA activity was also found using linear formamide-sucrose gradients. (2) Determination of both total mRNA activity and ovalbumin mRNA activity in the wheat germ cell-free translation assay revealed that 92% of the total peptides synthesized were specifically immunoprecipitable with an ovalbumin antiserum. (3) Analysis of the total peptides synthesizied in the wheat germ assay by sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated the presence of a single radioactive peak that corresponded exactly to a specifically immunoprecipitable ovalbumin standard. Thus, based on these observations ovalbumin mRNA appears to be greater than 95% pure. A preliminary estimation of the molecular weight of purified ovalbumin mRNA by formamide-containing sucrose gradients yielded a value of 520,000 or approximately 1600 nucleotides. This value was considerably less than the value of 900,000 obtained by gel electrophoresis under denaturing conditions. Analysis of the poly(A) content by a hybridization assay with (3H)poly(U) revealed the presence of a poly(A) region containing approximately 70 adenosine residues. Thus, the size of the ovalbumin mRNA is considerably greater than that required to code for a protein of 387 amino acids. The availability of large quantities of purified ovalbumin mRNA should now permit a more thorough analysis of its physical and chemical properties.

Animals↗
PubMed2001

Isolation of total RNA from pollens.

Isolation of total RNA from plant materials has been difficult, due to the presence of complex organic substances and the associated pigmentation. In fact, there is a dearth of standardized protocols for isolating total RNA from pollens. To find a simple and reliable method for isolating total RNA from pollen, four methods, viz. phenol/SDS (PS), guanidine HCl (GH), tri-reagent (TR), and modified SDS-betaME (SB) were tested with fresh pollen of Ricinus communis (procured at -70 degrees C) and pollen dried at 30-37 degrees C. The quality and quantity of RNA was superior for the material processed at -70 degrees C. SB gave the highest RNA yield (2.35 mg/g, OD260/280 >2.0), compared to other methods. The results obtained by the SB method were found to be comparable with the widely used tri-reagent method. This was validated with other pollens of Imperata cylindrica and Xanthium strumarium. The yield obtained from graded amounts of pollen was consistent with SB, compared to the TR method. The RNA isolated by SB gave good quality mRNA for synthesizing cDNA. The SDS-betaME method is simple, efficient, and uses less expensive reagents. Hence, we recommend the modified SDS-betaME method for isolating total RNA from pollens.

Guanidine↗
PubMed1981

RNA metabolism in isolated nuclei: processing and transport of immunoglobulin light chain sequences.

Transport of prelabeled RNA from isolated myeloma nuclei is studied using conditions that permit RNA synthesis. Cytosol and spermidine are not required to maintain nuclear stability and inhibited RNA release. Omission of ATP or GTP decreased release 25 to 40%. The stimulatory effect of ATP or GTP is not due to hydrolysis of the triphosphates by the nuclear envelope NTPase, since addition of quercetin (an inhibitor of this NTPase) has no effect on the quantity of RNA released. The size distribution and percentage of poly A-containing species released from nuclei incubated with or without ATP or the other rNTPs are identical. Hybridization analysis of nuclear RNA before the transport assay revealed mature and precursor k light chain mRNA sequences. Following the transport assay, a significant fraction of k mRNA precursors is chased into mature k mRNA which is found both in nuclear-retained and released RNA.

Animals↗
PubMed1977

Relative stability of alpha- and beta-globin messenger RNAs in homozygous beta+ thalassemia.

The relative concentrations of alpha-, beta-, and gamma-globin mRNA sequences were measured in bone marrow nuclear and cytoplasmic RNA and in RNA from peripheral blood reticulocytes of three patients with homozygous beta+ thalassemia. Our results suggest that the quantitative deficiency in beta-globin mRNA may arise because of abnormal metabolism of molecules containing beta mRNA sequences. Complementary DNAs specific for each of the globins were synthesized. Variable quantities of RNA were incubated to equilibrium with 3H-labeled alpha- and 32P-labeled beta- or gamma-enriched cDNA. We found for each of the patients that the alpha/beta mRNA sequence ratio was more nearly normal in the nuclear RNA than in either cytoplasmic or reticulocyte RNA. Conversely, gamma mRNA sequences were very low in the nucleus with an increase in the relative concentration in both cytoplasm and reticulocyte RNA. The thermal stability of nucleic acid duplexes formed between beta cDNA and nuclear RNA from one patient with beta+ thalassemia was equivalent to that of duplexes formed with normal nuclear RNA. Approximately equal amounts of thalassemic alpha and beta mRNA were retained by oligo(dT)-cellulose, indicating that the 3' poly(A) segment was present on both. Our results indicate that beta-globin mRNA, although grossly normal in structure, fails to accumulate in beta+ thalassemic erythroid cells in amounts equivalent to the mRNA for alpha-globin.

Bone Marrow↗
PubMed1985

Purification of functional RNA from human granulocytes.

The presence, in granulocytes, of high levels of nuclease activity makes it difficult to isolate intact RNA from these cells. We have developed a method that allows purification of functional RNA from normal granulocytes as determined by capability for reverse transcription and in vitro translation. We have shown that a considerable amount of ribonuclease activity remains in granulocyte lysates, even after the addition of heparin or vanadyl ribonucleoside complexes. RNA isolated from such lysates demonstrates only minimal binding to oligothymidylic-cellulose and does not serve as a template for reverse transcription or in vitro translation. However, the extraction of frozen granulocytes into phenol in the presence of both heparin and vanadyl ribonucleoside allows the purification of relatively large quantities of RNA, which serves as an excellent template for reverse transcription and in vitro translation. Purification of granulocyte RNA by this method will facilitate study of granulocyte gene expression.

Chromatography↗
PubMed1988

Preparation of both DNA and RNA for hybridization analysis from limiting quantities of lymphoid cells.

This report describes a method for preparing both DNA and RNA simultaneously from as few as 5 X 10(5) lymphoid cells. The method is suitable for cultured cells or any tissue from which a cell suspension can be prepared and for the small samples of purified cells obtained by fluorescence activated cell sorting. Cells are lysed with Nonidet P-40 and the nuclear and cytoplasmic fractions separated by centrifugation. Nuclei are embedded in low-gelling-temperature agarose and the proteinase K and restriction enzyme digestions performed whilst the DNA is immobilized in this form. Total RNA is prepared from the cytoplasmic fraction. This method is simple but reliable and is therefore particularly useful for preparing and analyzing the DNA and RNA from multiple samples when material is limited.

Animals↗
PubMed1993

In vitro transcription of transfer RNAs with 3'-end modifications.

It is difficult to modify the 3'-end of RNA transcribed in vitro from recombinant plasmid or phagemid DNA because of the need to retain a restriction endonuclease recognition site at the downstream end of the template in order to linearize the DNA before transcription. To avoid limitations on the 3'-sequence of RNA transcripts, we have modified a template-containing phagemid by inserting a FokI site outside the RNA gene sequence, positioned to cleave the template DNA to yield the desired 3' terminus. We demonstrate that this phagemid is an efficient template for tRNA transcription and use it to prepare mutants of Escherichia coli tRNA(Val) with modifications at the 3'-CCA end. Phagemids containing a FokI site should be suitable for in vitro transcription of large quantities of RNA of any length and with an unlimited variety of 3'-terminal sequences.

Base Sequence↗
PubMed1977

Messenger RNA for renal phosphoenolpyruvate carboxykinase (GTP). Its translation in a heterologous cell-free system and its regulation by glucocorticoids and by changes in acid-base balance.

A translational assay was used to measure the level of mRNA coding for phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) in the rat kidney in various conditions in which the enzyme is induced. RNA extracted from whole kidneys was chromatographed on oligo(dT)-cellulose to select poly(A)-containing RNA. This crude mRNA preparation was able to stimulate amino acid incorporation into protein in a cell-free system containing an extract of wheat germ. Phosphoenolpyruvate carboxykinase could be detected among the polypeptides synthesized and quantitated by immunoprecipitation with a monospecific antibody followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amount of enzyme synthesized was proportional to the quantity of RNA added. The level of mRNA coding for phosphoenolpyruvate carboxykinase is increased 3-fold 6 h after triamcinolone injection. Translatable enzyme mRNA also increases 3-fold within 6 h of the onset of metabolic acidosis caused by an ammonium chloride load. In both cases, the increase in functional mRNA is commensurate with the stimulation of enzyme synthesis measured in vivo. Glucocorticoid administration and acidosis cause additive increases in the level of translatable phosphoenolpyruvate carboxykinase mRNA. The inductive effect of acidosis is preserved in the absence of the adrenals, hypophysis, thyroid, and parathyroids.

Acid-Base Equilibrium↗
PubMed2005

Expression profiling using human tissues in combination with RNA amplification and microarray analysis: assessment of Langerhans cell histiocytosis.

Advances in molecular genetics have led to sequencing of the human genome, and expression data is becoming available for many diverse tissues throughout the body, allowing for exciting hypothesis testing of critical concepts such as development, differentiation, homeostasis, and ultimately, disease pathogenesis. At present, an optimal methodology to assess gene expression is to evaluate single cells, either identified physiologically in living preparations, or by immunocytochemical or histochemical procedures in fixed cells in vitro or in vivo. Unfortunately, the quantity of RNA harvested from a single cell is not sufficient for standard RNA extraction methods. Therefore, exponential polymerase-chain reaction (PCR) based analyses, and linear RNA amplification including amplified antisense (aRNA) RNA amplification and a newly developed terminal continuation (TC) RNA amplification methodology have been used in combination with microdissection procedures such as laser capture microdissection (LCM) to enable the use of microarray platforms within individual populations of cells obtained from a variety of human tissue sources such as biopsy-derived samples {including Langerhans cell histiocytosis (LCH)} as well as postmortem brain samples for high throughput expression profiling and related downstream genetic analyses.

Gene Expression Profiling↗
PubMed1992

Detection of Junin virus by the polymerase chain reaction.

Argentine hemorrhagic fever is an often fatal human disease caused by Junin virus, an RNA-containing virus and member of the Arenavirus family. This virus was detected in vitro by the polymerase chain reaction (PCR) procedure. A pair of Junin virus-specific PCR DNA oligonucleotide primers and an oligonucleotide probe were designed from a known portion of the viral RNA sequence. RNA was isolated from Junin virus-infected monkey kidney cells and used to produce complementary DNA (cDNA) by reverse transcription. A DNA segment, 151 +/- 24 bp long, was amplified from this cDNA and characterized by agarose gel electrophoresis and Southern blot hybridization with the Junin virus-specific DNA probe. Sensitivity experiments showed that Junin virus could be detected with nanogram quantities of RNA isolated from virus-infected cells. The rapid and sensitive assay described here may contribute towards the development of a procedure for the early diagnosis of Argentine hemorrhagic fever.

Arenaviruses, New World↗
PubMed1994

In vitro characterization of an internal ribosomal entry site (IRES) present within the 5' nontranslated region of hepatitis A virus RNA: comparison with the IRES of encephalomyocarditis virus.

The lengthy 5' nontranslated region (5'NTR) of hepatitis A virus (HAV) forms a highly ordered secondary structure, which has been suggested to play an important role in controlling viral translation by allowing for translation initiation by internal ribosome entry. To test this hypothesis, synthetic bicistronic RNAs, with all or part of the HAV 5'NTR in the intercistronic space, were translated in rabbit reticulocyte lysates. In the presence of an upstream cistron designed to block ribosomal scanning, the HAV 5'NTR was capable of directing the internal initiation of translation, confirming the presence of an internal ribosome entry site (IRES). Analysis of various deletion mutants demonstrated that the 5' border of the IRES is located between nucleotides 151 and 257, while the 3' border extends to the 3' end of the 5'NTR, between nucleotide 695 and the first initiation codon at 735. Except for a segment between bases 638 and 694, deletion of stem-loop structures between bases 151 and the 3' end of the 5'NTR inhibited or abolished translation. The addition of a 5' cap structure (m7GpppN) to monocistronic or bicistronic transcripts decreased the translation of a reporter gene downstream of the HAV 5'NTR but enhanced translation of the upstream cistron in bicistronic transcripts. This finding indicates that a 5' cap structure is inhibitory to HAV IRES-directed translation initiation and that the cap structure and the HAV IRES probably compete for the same limiting translation factors. The efficiency with which monocistronic constructs containing the HAV 5'NTR directed translation in reticulocyte lysates was compared with results for monocistronic constructs containing the IRES of the more rapidly growing encephalomyocarditis virus (EMCV). These results indicated that the HAV 5'NTR was more than 25-fold less active than the EMCV IRES in producing translation product. HAV 5'NTR-directed translation was inhibited by the presence of a one-fifth molar quantity of RNA containing the EMCV IRES, while a fivefold molar excess of the HAV 5'NTR did not inhibit EMCV IRES-directed translation. The relatively weak activity of the HAV IRES may thus be due to a reduced affinity for cellular translation factors which are present in limiting quantities in rabbit reticulocyte lysate.

Animals↗