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PubMed1990

Expression of six mitochondrial genes during Drosophila oogenesis: analysis by in situ hybridization.

We have done a comparative analysis of RNA from six mitochondrial genes (rDNA, ND2, COI, COIII, ND4-ND5, Cyt b) during Drosophila oogenesis, using in situ hybridization. This study showed the same variation for each of these transcripts, which is similar to that obtained with the total mitochondrial RNA (Tourmente et al. (1990) Biol. Cell 60, 119-127). A constant RNA density until stage 9, followed by a rapid decline, was observed in follicle and nurse cells. These results confirm those previously obtained (Tourmente et al., (1990) Biol. Cell 60, 119-127), in favor of the existence of a correlation between the mtRNA level and the cell volume and/or the nuclear DNA content, and suggest a global extra-mitochondrial, transcriptional control mechanism. We also show that the relative proportions of the different RNA are similar, whatever the stage and cell type examined, even though the total mtRNA quantity is different. They are comparable to those previously obtained by Northern analysis of Drosophila embryos (Berthier et al. (1986) Nucleic Acids Res. 14, 1400-1412, suggesting a posttranscriptional control independent of the cell type. Surprisingly, we have detected an extra-mitochondrial hybridization for COIII, both in light and electron microscopy. Northern analysis of poly(A)+RNA from ovaries or cultured cells revealed an 1.7 kb extra-mitochondrial RNA, which is probably of nuclear origin.

Animals↗
PubMed1975

[Attempts at localisation and evaluation of the RNA amounts in the tail and the cytoplasmic droplet of bull spermatozoa].

The RNA has been located and proportioned by a spectrophotometric method which absorbs the ultra-violet light. The technique used allows us to execute punctual dosage into the spermatozoon's tail. Each of these punctual dosages makes us realise the RNA localisation; and the sum of these dosages makes us realise the total quantity which is contained in one spermatozoon. The signification of the collected data is debated. A comparison has been made with chemical methods. These methods give the advantage to obtain valuations on large quantities of spermatozoa. But these valuations are always global value, and the individual value is always a mean's expression. Without being dull, the microspectophotometry is slow and brings about to effect on much restricted numbers, but the data are all individual measurements, (and we can even obtain punctual measurements in the range of 400 to 500 for the tail only. So that we can interprete these individual measurements and make very interesting comparisons from cell to cell. The possibilities are therefore very large and they complete fortunately the chemical methods.

Male↗
PubMed2006

Nonisotopic detection of microRNA using digoxigenin labeled RNA probes.

MicroRNAs (miRNAs) are an important class of endogenously derived, small approximately 22 nucleotide noncoding regulatory RNAs that have recently become implicated in development, cell regulation and cancers of various tissues. Here we report a nonisotopic Northern analysis method for miRNA detection using 3'-digoxigenin (DIG)-labeled RNA oligo probes. Northern blot analysis was performed using miRNA or total RNA fractions extracted from human leukemic cell lines, and blots were hybridized with either 32P- or DIG-labeled RNA probe for miR-181, miR-155 or miR-16. A labeled probe for U6 small nuclear RNA served as an internal control. The use of DIG-labeled RNA probes was equally sensitive compared to 32P-labeled probes in detecting miRNA quantities as low as 50 ng. The ability to use nonisotopic methods and yet obtain sensitive and reliable results offers an advantage to investigators who prefer to avoid isotopes.

Blotting, Northern↗
PubMed1975

A protein from polymorphonuclear leukocytes (LEM) which affects the rate of hepatic amino acid transport and synthesis of acute-phase globulins.

A proteinaceous secretion from phagocytizing polymorphonuclear leukocytes, termed "leukocytic endogenous mediator" (LEM), has been shown to have marked effects on hepatic amino acid transport and RNA and protein synthesis. A single injection of LEM results in a marked accumulation of labeled nonmetabolizable model amino acids in the liver of normal rats. The LEM-stimulated uptake of amino acids by liver was observed in adrenalectomized, hypophysectomized, thyroidectomized, or diabetic rats and could not be duplicated by pharmacological doses of a large variety of hormones. In addition, LEM stimulated an increased uptake of alpha-aminoisobutyric acid by isolated livers during their perfusion in vitro. LEM also stimulated an increased incorporation of orotic acid into hepatic RNA of intact rats, especially into the bound ribosomal fraction. This increased synthesis of RNA preceded an enhanced hepatic production of a number of the acute-phase plasma globulins. LEM did not stimulate the adenylate cyclase-cAMP system in liver and was not found to utilize this system as a second messenger. Thus, the effects of LEM in stimulating hepatic amino acid transport appear to be direct, without mediation by other hormones, and to be independent of cAMP. On the other hand, the ability of LEM to stimulate RNA and acute phase globulin synthesis in liver may require the presence of physiological quantities of hormones such as adrenal corticoids.

Adenylyl Cyclases↗
PubMed1985

Activation of the c-K-ras oncogene in a human pancreas carcinoma.

The human pancreas carcinoma cell line T3M-4 contains activated c-Kirsten (K)-ras oncogene detectable by the DNA-mediated gene transfer technique using NIH/3T3 cells. DNA fragments containing coding lesions have been cloned, and nucleotide sequence analysis suggests that the T3M-4 oncogene has been activated by a single nucleotide transition from A to C in the second exon, which results in the substitution of histidine for glutamine in coden 61 of the predicted amino acid sequence. The quantity analysis of c-K-ras oncogene in the DNA and RNA of T3M-4 cells revealed that the c-K-ras gene was amplified and overexpressed in T3M-4 cells. These findings indicate that the T3M-4 c-K-ras oncogene is activated by different mutational events.

Amino Acid Sequence↗
PubMed1973

Guanosine 5'-diphosphate, 3'-diphosphate: assignment of structure by 13C nuclear magnetic resonance spectroscopy.

Guanosine tetraphosphate, recently discovered to mediate the regulatory relationship between protein synthesis and RNA accumulation in various bacteria, has been synthesized in vitro in large quantities and analyzed by natural-abundance (13)C nuclear magnetic resonance spectroscopy in order to confirm its structure and establish the positions of phosphate attachment. These studies have established its structure as guanosine 5'-diphosphate, 3'-diphosphate.

Carbon Isotopes↗
PubMed1999

Tobacco mosaic virus and the virescence of biotechnology.

There is a growing realization that a modern combination of molecular biology and agriculture will provide a photosynthetic basis for the biosynthesis of an increasing variety of complex and valuable molecules. This 'greening' of biotechnology may impact on the global environment in many beneficial ways, but will perhaps have its most significant impact on human health. In the past decade, the capacity to use plants as an expanded source of therapeutics has grown through the accelerated development of effective viral transfection vectors for gene transfer to cultivated crops. Recombinant vectors based on tobacco mosaic virus (TMV) and other members of the Tobamovirus genus are now used to transfect commercially meaningful quantities of plant biomass cultivated in enclosed greenhouses and multiacre fields. Viral RNA promoters are effectively manipulated for the synthesis of recombinant messenger RNAs in whole plants. Chimeric plant virus and virus-like particles are designed for peptide production and display from recombinant structural protein-gene fusions. Gene functions are assessed and modified by either virus-mediated expression or cytosolic inhibition of expression at the RNA level. Recombinant virus populations, propagated by inoculating plants with infectious RNA transcripts or recombinant virions, have proved to be genetically stable over product-manufacturing cycles. Large volumes of highly purified protein products isolated from transfected foliage conform reproducibly to the specifications required for well-characterized biologics. In some cases, they exceed the specific activities of molecules purified from alternative recombinant and native sources. The resulting products are then formulated according to the developing national regulatory guidelines appropriate for agriculture-based manufacturing. Each of these innovations was first realized by researchers using clones of tobamovirus genes and recombinant genomes. This progress is founded on the heritage of a century of fundamental TMV research.

Genetic Vectors↗
PubMed2005

TmaDB: a repository for tissue microarray data.

BACKGROUND: Tissue microarray (TMA) technology has been developed to facilitate large, genome-scale molecular pathology studies. This technique provides a high-throughput method for analyzing a large cohort of clinical specimens in a single experiment thereby permitting the parallel analysis of molecular alterations (at the DNA, RNA, or protein level) in thousands of tissue specimens. As a vast quantity of data can be generated in a single TMA experiment a systematic approach is required for the storage and analysis of such data. DESCRIPTION: To analyse TMA output a relational database (known as TmaDB) has been developed to collate all aspects of information relating to TMAs. These data include the TMA construction protocol, experimental protocol and results from the various immunocytological and histochemical staining experiments including the scanned images for each of the TMA cores. Furthermore the database contains pathological information associated with each of the specimens on the TMA slide, the location of the various TMAs and the individual specimen blocks (from which cores were taken) in the laboratory and their current status i.e. if they can be sectioned into further slides or if they are exhausted. TmaDB has been designed to incorporate and extend many of the published common data elements and the XML format for TMA experiments and is therefore compatible with the TMA data exchange specifications developed by the Association for Pathology Informatics community. Finally the design of the database is made flexible such that TMA experiments from several types of cancer can be stored in a single database, which incorporates the national minimum data set required for pathology reports supported by the Royal College of Pathologists (UK). CONCLUSION: TmaDB will provide a comprehensive repository for TMA data such that a large number of results from the numerous immunostaining experiments can be efficiently compared for each of the TMA cores. This will allow a systematic, large-scale comparison of tumour samples to facilitate the identification of gene products of clinical importance such as therapeutic or prognostic markers. In addition this work will contribute to the establishment of a standard for reporting TMA data analogous to MIAME in the description of microarray data.

Data Interpretation, Statistical↗
PubMed1974

Abolition of natural tolerance and the influence of the chemical allergen beryllium on autoimmune processes.

PULMONARY BERYLLIOSIS, A CHRONIC ALLERGIC PNEUMOSCLEROSIS RESULTING FROM INHALATION OF BERYLLIUM COMPOUNDS, MAY OCCUR IN TWO CLINICAL FORMS: interstitial, which is mild, slowly progressive, and presents moderate skin reactions of delayed-type hypersensitivity to haptens; and granulomatous, which is rapidly progressive after a short latent period and presents systemic lesions and significant allergic skin reactions. The investigation of experimental berylliosis in rats has revealed some factors that could lead to the abolition of natural tolerance. Six new beryllium-containing autoantigens, two of which accumulated in different tissues, were identified in the lung nucleoproteins accompanying a partial loss of normal tissue and serum antigens. Antibodies to the new antigens and to normal lung tissue (in smaller amounts) were found in the sera of rats with experimental berylliosis, as well as in patients. The patients with granulomatous berylliosis also had antibodies to DNA, RNA, and extracts of normal homologous heart, spleen, liver, and thyroid in quantities that correlated with the clinical picture and with the effectiveness of glucocorticoid therapy. The fact that natural tolerance mechanisms were interrupted in all cases and autoimmune granulomatous berylliosis developed only in some patients led to the assumption that the mechanism was effective under the influence of additional endogenous factors; diseases producing an accumulation of autoantibodies could provide the stimulus for the appearance of these factors.

Animals↗
PubMed1996

Homozygosity for two point mutations in the lipoprotein lipase (LPL) gene in a patient with familial LPL deficiency: LPL(Asp9-->Asn, Tyr262-->His).

Familial lipoprotein lipase (LPL) deficiency is an inherited disorder of lipoprotein metabolism characterized by hypertriglyceridemia and recurrent episodes of abdominal pain and pancreatitis. We have studied the genetic basis of LPL deficiency in a 62-year-old black male with undetectable pre- and post-heparin plasma LPL mass and activity, DNA sequence analysis of the patient's LPL cDNA and gene as well as digestion with Bcl I and Asu I revealed that the proband is a homozygote for two separate gene defects. One mutation changed a G to an A, resulting in the conversion of amino acid 9 of the mature protein, aspartic acid (GAC), to asparagine (AAC). The second substitution, a C for a T, replaced tyrosine (TAC) at residue 262 with histidine (CAC). Northern blot analysis of monocyte-derived macrophage RNA demonstrated the presence of LPL mRNA of approximately normal size and quantity when compared to control. Expression of both mutations separately (pCMV-9 and pCMV-262) or in combination (pCMV-9+262) in human embryonal kidney-293 cells demonstrated that LPL-9 had approximately 80% the specific activity of wild type LPL, but LPL-262 and LPL-9+262 had no enzymic activity, thus establishing the functional significance of the LPL-262 defect. Despite an absolute deficiency of LPL mass and activity demonstrated by analysis of patient post-heparin plasma, in vitro expression of both LPL mutants was normal, suggesting that the absence of LPL in patient post-heparin plasma was a result of altered in vivo processing. Analysis of the heparin binding properties of the mutant enzymes by heparin-Sepharose affinity chromatography indicated that most of the LPL-262 mass was present in an inactive peak, which like the normal LPL monomer, eluted at 0.8 M NaCl. Thus, the Tyr262 --> His mutation may alter the stability of the LPL dimer, leading to the formation of inactive LPL-262 monomer which exhibits reduced heparin affinity. Based on these results, we propose that, in vivo, enhanced formation of LPL-9+262 monomer leads to abnormal binding of the mutant lipase to endothelial glycosaminoglycans ultimately resulting in enhanced catabolism of the mutant enzyme and lower enzyme mass in post-heparin plasma.

Amino Acid Sequence↗
PubMed2001

Natural minus-strand RNAs of alfalfa mosaic virus as in vitro templates for viral RNA polymerase. 3'-terminal non-coded guanosine and coat protein are insufficient factors for full-size plus-strand synthesis.

Replication complexes of alfalfa mosaic virus produce in vivo large quantities of plus-strand RNAs, but this production is fully dependent on the presence of coat protein. In order to study this process of RNA-dependent and coat protein-regulated RNA synthesis we have isolated the three natural minus-strand RNAs (containing any posttranscriptional modification that might have occurred) and have tested them for coat protein binding sites and template activity in an in vitro system with the viral RNA polymerase. The enzyme was prepared by an advanced isolation procedure. All three minus strands had a single non-coded G at their 3' terminus. They were not able to withdraw coat protein subunits from virions as free virion RNAs do. No sites protected by coat protein against ribonuclease T1 degradation were found. Two large T1 oligonucleotides from minus RNA 1 and one from minus RNA 3 were bound by coat protein to Millipore filters. Except for minus RNA 3 which caused a minute amount of full-size plus strand to be synthesized, the minus strands did not function as templates for full-size complementary strands. On the other hand, they gave rise to a number of well-defined shorter products, the synthesis of which was stimulated by the addition of coat protein. These products could not be elongated by a chase treatment and were probably the result of internal initiations. It is concluded that, although posttranscriptional modifications of the template and the presence of coat protein may be necessary factors for plus-strand RNA synthesis, they are certainly not sufficient. Our purified in vitro system needs further sophistication.

3' Untranslated Regions↗
PubMed1998

Induction of cytokine messenger RNA transcripts in mouse macrophages by Listeria monocytogenes isolated from channel catfish.

OBJECTIVE: To determine whether differences exist in induction and quantity of tumor necrosis factor alpha (TNF-alpha), interleukin (IL)1 beta, and IL-10 mRNA transcripts when mouse J774A.1 macrophages are infected with Listeria monocytogenes, including 2 isolates originating from channel catfish, the wild-type virulent (EGD) strain, and a nonhemolytic strain (ATCC 15313). SAMPLES: Listeria monocytogenes isolates from kidneys or fillets of channel catfish were used to stimulate cytokine production from mouse macrophages. The RNA from the infected macrophages was collected. PROCEDURE: Four hours after infection with L monocytogenes, total cellular RNA was extracted from the J774A.1 cells and reversed transcribed to cDNA, which was amplified, using specific primers for TNF-alpha, IL-1 beta, or IL-10. The specific amplified DNA fragments were detected on polyacrylamide gels and quantified, using a reverse transcription polymerase chain reaction (PCR)-mediated ELISA. RESULTS: The wild-type hemolytic EGD strain and the 2 hemolytic catfish isolates of L monocytogenes induced higher amounts of TNF-alpha-, IL-1 beta-, and IL-10-specific mRNA in J774A.1 cells than did the nonhemolytic strain. CONCLUSIONS: Hemolysin-associated induction of TNF-alpha, IL-1 beta, and IL-10 cytokines may be related to survival and replication of L monocytogenes in macrophages. It also suggests that the PCR-mediated ELISA procedure is a sensitive test to quantify cytokines from cell cultures.

Animals↗
PubMed1988

A new sensitive non-isotopic method using sulfonated probes to detect picogram quantities of specific DNA sequences on blot hybridization.

The use of non-radioactive systems to detect target DNA or RNA displays many advantages such as safe manipulation, potential use in non-specialized scientific area and prolonged lifetime of the probes (one year or more). We here describe a method we have improved and optimized using sulfonated DNA probes for hybridization on dot and Southern blots. Sulfonation is an easy chemical modification procedure which does not require enzymatic coctail as does nick-translation. Sensitivity of this method has been particularly improved by using a new blocking solution, containing heparin, which allows easy and fast detection of picogram quantities of DNA. This method allows the use of nitrocellulose as well as nylon membranes with very low background. Equal resolution is obtained in comparative experiments involving both sulfonated and 32P-radiolabelled probes. Single copy gene sequences are readily detected in nuclear DNA. These results allow the use of this procedure for restriction fragment length polymorphism (RFLP) studies.

Base Sequence↗
PubMed1986

Detection of vasoactive intestinal peptide-encoding messenger ribonucleic acid in the rat ovaries.

Vasoactive intestinal peptide (VIP) has recently been detected in rat ovaries and has been shown to stimulate steroidogenesis by cultured rat granulosa cells. In this study we investigated whether the VIP-messenger RNA (mRNA) can be detected in the ovaries, thus suggesting local synthesis of the peptide. To study VIP-gene expression, a sensitive RNA detection assay which uses in vitro transcribed RNA probes corresponding to specific exons of the VIP gene was developed. Using this method, an approximately 2000-base RNA band containing the coding sequences for VIP was detected in rat ovaries. This RNA also contains the coding sequences for the VIP-related peptide (peptide-histidine-methionine). An identical VIP-encoding RNA was previously identified in the rat cerebral cortex. However, the VIP-mRNA quantity in the cortex was 12-fold-higher as compared to the ovaries. These results may reflect the differences in VIP concentration in the two organs. The finding of VIP-encoding mRNA in the rat ovaries suggests a local synthesis of VIP in the ovaries.

Animals↗
PubMed1976

[Age and dynamics of protein and RNA concentration in sheep oocytes].

There exists a definite age rhythm of the amount of RNA and protein in Graaf's vesicle oocytes in the postnatal life of the sheep. The lowest quantities of these components were found in the prepubertal lambs; maximum was recorded in 1,5-2,5 year ewes and later the amount of these components decreased and the oocyte of 8,5 year ewes contained 24% less general protein and 69% less RNA than 1,5-2,5 year ewes. As the oocyte grows the amounts of the protein and RNA change. A primordial follicle oocyte contains 250 +/- 15 rel. units of general protein while a Graff's follicle oocytes has three times more (767 +/- 67 rel. unit). The corresponding data for the cytoplasm RNA are: 87,5 +/- 5 rel. units and 163 +/- 18 rel. units which means a 2 times increase.

Age Factors↗
PubMed1992

Preparation of 13C and 15N labelled RNAs for heteronuclear multi-dimensional NMR studies.

A procedure is described for the efficient preparation of isotopically enriched RNAs of defined sequence. Uniformly labelled nucleotide 5'triphosphates (NTPs) were prepared from E.coli grown on 13C and/or 15N isotopically enriched media. These procedures routinely yield 180 mumoles of labelled NTPs per gram of 13C enriched glucose. The labelled NTPs were then used to synthesize RNA oligomers by in vitro transcription. Several 13C and/or 15N labelled RNAs have been synthesized for the sequence r(GGCGCUUGCGUC). Under conditions of high salt or low salt, this RNA forms either a symmetrical duplex with two U.U base pairs or a hairpin containing a CUUG loop respectively. These procedures were used to synthesize uniformly labelled RNAs and a RNA labelled only on the G and C residues. The ability to generate milligram quantities of isotopically labelled RNAs allows application of multi-dimensional heteronuclear magnetic resonance experiments that enormously simplify the resonance assignment and solution structure determination of RNAs. Examples of several such heteronuclear NMR experiments are shown.

Base Sequence↗
PubMed1987

Isolation and characterization of nucleoprotein assembly intermediates of tobacco mosaic virus.

During assembly of tobacco mosaic virus from pure RNA and 20S capsid protein aggregates under conditions where protein is limiting, partially assembled intermediates of specific sizes accumulate; these were isolated on sucrose density gradients. The earliest intermediate found in substantial quantity sedimented at 56 S and was shown, by measurement of its buoyant density and of the length of the RNA segment protected by the capsid protein from nuclease digestion, to consist of RNA that is 13% encapsidated (corresponding to a rod length of about 39 nm); the next intermediate sediments at 78 S and is 18% encapsidated (corresponding to a rod length of about 54 nm). Studies of the distribution of intermediates at various input ratios of protein/RNA indicated that their accumulation results from decreases in the rate constants for protein binding that are local to specific points in the course of encapsidation. After extensive nuclease digestion, the RNA still associated with the first intermediate was shown to include a portion that is unencapsidated. This segment of the RNA may be a region of stable secondary that confers the nuclease resistance despite the lack of protection by capsid protein. Such RNA secondary structure, if it exists, would also cause the accumulation of intermediates by imposing an energy barrier to subsequent rod elongation.

Capsid↗