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PubMed1990

Expression of glial fibrillary acidic protein and neurofilament mRNA in gliosis induced by experimental autoimmune encephalomyelitis.

We have previously shown that the content of glial fibrillary acidic protein (GFAP) gradually increases in the spinal cord of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE), reaching a level 1.5-2 times greater than that in controls by 35 days postimmunization (dpi). We report here that the increase in GFAP mRNA level followed a completely different time course and reached higher levels relative to controls than did that of the protein. Total RNA was isolated using a modified version of current methods using phenol/chloroform extractions to ensure optimal recovery from spinal cord. Control animals yielded 323 +/- 35 micrograms (mean +/- SD; n = 34) of total RNA/spinal cord throughout the experimental period. EAE animals contained up to three times as much total RNA during 11-14 dpi, a finding largely reflecting the infiltration of inflammatory cells. By 65 dpi, total RNA levels closely approached control values. As early as 10 dpi, increased amounts of GFAP mRNA were detected in EAE animals relative to controls. During 11-14 dpi, GFAP mRNA levels reached six- to eightfold greater than values in controls and then slowly declined throughout the remainder of the time course, with a fourfold increase still detected at 65 dpi. However, coinciding with the height of inflammation and clinical signs at 12 dpi, the GFAP mRNA content dropped to approximately 50% of the level at 11 dpi but rose again at 13 dpi. This dip was mirrored by a similar decrease in neurofilament mRNA content, but otherwise the level of this message remained relatively constant and equal to that in controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

RT-PCR quantification of AHR, ARNT, GR, and CYP1A1 mRNA in craniofacial tissues of embryonic mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and hydrocortisone.

C57BL/6N mouse embryos exposed to hydrocortisone (HC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) develop cleft palate. An interaction between these agents produces clefts at doses which alone are not teratogenic. The glucocorticoid receptor (GR) and dioxin receptor (AhR) mediated these responses and their gene expression was altered by TCDD and/or HC in palates examined on gestation day (GD) 14 by Northern blot analysis and in situ hybridization. The present study quantifies AhR, AhR nuclear translocator (ARNT), and GR mRNA at 4, 12, 24, and 48 h after exposure (time 0 = dose administration at 8 A.M. on gestation day 12) on GD12 to TCDD (24 micrograms/kg), HC (100 mg/kg) or HC (25 mg/kg) + TCDD (3 micrograms/kg). The induction of CYP1A1 mRNA was also quantified at 2, 4, 6, 12, 24, and 48 h for control and TCDD-exposed samples. Total RNA was prepared from midfacial tissue of 4-6 embryos/litter at each time and dose. An RNA internal standard (IS) for each gene was synthesized, which included the gene's primer sequences separated by a pUC19 plasmid sequence. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on total RNA + IS using a range of 5-7 IS concentrations across a constant level of total RNA. PCR products were separated in gels (mRNA and IS-amplified sequences differed by 30-50 bases), ethidium bromide-stained, imaged (Hamamatsu Photonics Systems, Bridgewater, NJ), and quantified with NIH Image. CYP1A1 mRNA was significantly induced in the TCDD-exposed samples at all time points examined (p = 0.005 at 2 h and 0.001 after 2 h). During palatal shelf outgrowth on GD12, AhR mRNA levels increased significantly and this was not affected by treatment with TCDD or HC + TCDD. A significant increase in GR was detected at 24 h (p < 0.05) and this was unaffected by any of the exposures. Expression of ARNT increased at 12 h (p < 0.001); however, treatment with HC or HC + TCDD blocked this increase (p < 0.05). At 24 h, the TCDD-treated embryos had significantly lower ARNT mRNA compared with controls (p < 0.001). The relative overall expression level of the genes was AhR > ARNT > GR. Within individuals, expression of AhR and/or ARNT was highly correlated with GR level. In conclusion, CYP1A1 mRNA was expressed in developing craniofacial tissue and was highly induced by TCDD exposure. AhR, ARNT, and GR mRNA are upregulated in early palatogenesis, although not on the same schedule. The TCDD-induced decrease in ARNT at 24 h after dosing and the HC and HC + TCDD-induced delay in upregulation of ARNT may affect the dynamics of heterodimer formation between AhR and ARNT. The changes in ARNT mRNA level could also affect availability of this transcriptional regulator to interact with other potential partners, and these effects, separately or in combination, may be involved in disruption of normal embryonic development.

Animals↗
PubMed1993

Cloning of the cDNA coding for 14 kDa group II phospholipase A2 from rat liver.

The amino acid sequence of rat liver phospholipase A2 was partially elucidated using peptide fragments generated by enzymatic or chemical cleavage. Based on this sequence information, two oligonucleotide probes were constructed which were applied in a polymerase chain reaction on cDNA generated from rat liver total RNA. This resulted in cloning of the cDNA corresponding to the coding region of the mature phospholipase A2. The deduced amino acid sequence showed the enzyme belongs to the group II phospholipases, and is almost completely identical to rat platelet and spleen membrane-associated phospholipase A2. However, in the cDNA isolated one codon was different as compared to the platelet and spleen enzymes, resulting in the substitution of Ala94 by Arg94 in the liver enzyme. In Northern blot analyses the mRNA for rat group II phospholipase A2 could not be detected in rat liver, neither in total RNA nor in poly(A)+ RNA. However, a polymerase chain reaction using total RNA originating from freshly isolated hepatocytes resulted in the amplification of the described phospholipase A2 cDNA. This indicates that group II PLA2 mRNA is present in these cells, but presumably at very low abundance. The observed increase in rat group II phospholipase A2 secretion in rat mesangial cells upon stimulation with interleukin-1 beta (Pfeilschifter et al. (1989), Biochem. Biophys. Res. Commun. 159, 385-394) was shown to be accompanied by an increased transcription of the rat group II phospholipase A2 gene, indicating interleukin exerts its effect via increased phospholipase A2 mRNA synthesis. Based on Northern blot analyses of stimulated rat mesangial cells, the size of the mRNA for rat group II phospholipase A2 was determined to be 0.9 kb.

Amino Acid Sequence↗
PubMed1976

Relationship of senescence of corn (Zea mays L.) stalk pith, cob parenchyma and first developed leaf tissues to RNA content and synthesis.

In stalk parenchyma tissue of WF9 X 38-11 single corn (Zea mays L.), there was a per cell increase in RNA synthesis and a slight increase in total RNA as cells became older. In cob parenchyma tissue of WF9 X 38-11 single cross corn, both total RNA content and RNA synthesis per cell decreased with the age of cells. In first developed leaf tissue of FR43 X FR14A single cross corn, RNA synthesis increased steadily but only slightly over its life span. The pattern of RNA synthesis and destruction in senescing leaf tissue of seedlings and two sources of parenchyma tissue of maturing plants appeared to differ.

Cell Survival↗
PubMed1994

Relative quantitative analysis of glucagon receptor mRNA in rat tissues.

Total RNA prepared from nine rat tissues were analyzed for their content in glucagon receptor mRNA by two independent hybridization approaches: (1) simple dot blot analysis using labelled oligodeoxynucleotide; (2) highly specific RNase protection assay using labelled antisense RNA. Hybridization signal was quantified by laser densitometric scanning of autoradiographies. Results were expressed for each method relative to the liver content (100%) for either a constant amount of total RNA or for a constant amount of beta-actin mRNA. We obtained similar relative values of glucagon receptor mRNA per constant amount of total RNA by the two hybridization methods: in liver (100 and 100), in kidney (38 and 34), and in heart (12 and 11). The glucagon receptor mRNA was overestimated by the less specific dot assays, in adrenal glands (21 versus 10) and in adipose tissues (24 versus 5). In the stomach, brain, duodenum and lung, the signal was equal to or below the reliable quantification limit. Reverse transcription and polymerase chain reaction (RT-PCR) of glucagon receptor mRNA with limited cycle number were performed using two sets of primers: the first set amplified a single band at the 3' coding end, and the second, 3-6 bands at the 5' coding end, revealing tissue-specific polymorphism. RT-PCR data confirmed the presence of glucagon receptor mRNA in liver, kidney, heart, adrenal glands and adipose tissue, and allowed the detection of a very low amount of glucagon receptor mRNA in the stomach, the duodenum and brain but not in the lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed2004

Expression of transthyretin and retinol binding protein mRNAs and secretion of transthyretin by cultured monkey retinal pigment epithelium.

PURPOSE: To document the expression of mRNA for transthyretin (TTR) and retinol binding protein (RBP) in native and cultured Rhesus monkey retinal pigmented epithelium (RPE); to compare mRNA transcripts for these two proteins expressed in RPE with those found in whole monkey liver and brain; to demonstrate the secretion of TTR by RPE during short-term maintenance in a protein-free, defined medium, as a manifestation of the differentiated state of these cells in vitro. METHODS: Total RNA was isolated from cultured RPE in first passage, after incubation for eight days in defined, protein-free medium. Conditioned medium was collected for western analysis at this time. Total RNA was also extracted from RPE/choroid freshly dissected from monkey eyes. Using cDNA probes for human TTR and RBP, northern analysis was performed on the total RNA from fresh and cultured RPE samples, together with poly(A+) mRNA purified from monkey liver and brain. RESULTS: Conditioned medium from RPE yielded TTR protein of the expected monomer subunit molecular size. The TTR secreted de novo from the cultured cells was detectable in the absence of biosynthetic labeling. With the exception of some extremely low abundance transcripts expressed in cultured RPE, all samples contained a single 900 bp transcript for TTR. Based on relative amounts of actual message, RPE ranks higher than liver in abundance of TTR mRNA. In contrast, both native monkey RPE and cultured RPE cells expressed comparatively low levels of mRNA for RBP. All samples displayed a single RBP mRNA transcript at 1100 bp. CONCLUSIONS: Our results indicate that TTR is a significant gene product of the RPE, and may be considered as a marker for a differentiated phenotype for these cells in culture. There is increased recognition of various forms of ocular pathology associated with mutations or other malfunctions involving TTR and RBP, warranting a greater understanding of mechanisms of transcriptional and translational control for these two proteins.

Animals↗
PubMed1988

Vitellogenin gene expression in male rainbow trout (Salmo gairdneri).

Estrogen has a marked effect on the expression of vitellogenin, the egg yolk precursor protein in the liver of egg-laying vertebrates. cDNA clones specific for trout vitellogenin mRNA have been used to study the expression of the vitellogenin genes in rainbow trout (Salmo gairdneri). The steady-state levels of vitellogenin mRNA in the liver of male rainbow trout were measured during primary and secondary stimulation with estradiol. The kinetics of induction in trout appear to be very similar to those seen in Xenopus and chicken in that a lag of approximately 2 days is observed in the accumulation of serum vitellogenin during primary induction. This lag is not observed during the secondary stimulation. The primary induction of vitellogenin mRNA in trout liver, using a single injection of estradiol (3 mg/kg body wt) results in a short-lived rise, reaching a maximum level of 260 ppm total RNA on Day 2. Using silastic implants of estradiol to induce a primary response produces a large increase in the steady-state level of vitellogenin mRNA which reaches a maximum of 2750 ppm total RNA on Day 10. During secondary stimulation, using silastic tubing the maximum level reached was 1200 ppm of the total RNA on Day 7, approximately half the level seen in the primary induction using the silastic implants. The difference in these two levels is due to an increase in the steady-state levels of rRNA, which appear to increase between Days 10 and 21 after the primary stimulation. These results demonstrate that the induction of vitellogenesis in the trout by estradiol involves changes in the steady-state levels of a number of different mRNA and rRNA sequences and resembles that seen in Xenopus and chicken.

Amino Acid Sequence↗
PubMed2003

Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice.

Reverse transcription followed by real-time quantitative polymerase chain reaction (RT Q-PCR) is useful for the systematic measurement of plant physiological changes in gene expression. The validity of using 18S rRNA and three housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase, actin, and tubulin, was tested as a reference of RT Q-PCR. Under various growth stages of etiolated seedlings, different cultivars, and various times after UV-irradiation treatment, expression level of 18S rRNA correlated with total RNA suggesting the uniformity of RT Q-PCR efficiencies among samples. Relative expressions of housekeeping genes varied among samples and independently of experimental conditions, up to two-fold, signifying generally constant fraction of mRNA in total RNA. Results indicate 18S rRNA was the most reliable reference gene for RT Q-PCR of total RNA.

Actins↗
PubMed1991

Rapid method for direct extraction of mRNA from seeded soils.

A protocol for direct extraction of mRNA from soil samples was developed. Soil samples (10 g) were washed twice with 120 mM phosphate buffer (pH 5.2). The lysis of cells, fixation of RNA, and hydrolysis of DNA were achieved by vigorously shaking the washed soil in a 4 M guanidine thiocyanate solution containing 25 mM sodium citrate, 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol. The pH of the homogenized mixture was adjusted with 2 M sodium acetate (pH 4.0); the mRNA was then extracted with phenol and chloroform. Total RNA was precipitated with isopropanol. This method extracts up to 17 mug of total RNA per g (wet weight) of soil containing 8.0 x 10 cells of Pseudomonas aeruginosa PU21, and mRNA has been detected in 160-ng total RNA fractions. This method has been used for the detection of mRNA transcribed from specific biodegradative genes, including the nah and mer operons, in contaminated soils. This extraction method can be completed within a few hours and has tremendous potential for ecological studies of in situ gene expression among soil microbiotas.

Journal Article↗
PubMed1994

Tissue specificity of rat mitochondrial dimethylglycine dehydrogenase expression.

Expression of mitochondrial dimethylglycine dehydrogenase (Me2GlyDH) was analysed in various tissues, liver cell types and developmental stages of the rat. Total RNA extracted from liver, spleen, brain, kidney, lung and heart was reverse-transcribed into cDNA and amplified with Me2GlyDH cDNA-specific oligonucleotides by PCR. Expression of the enzyme was observed mainly in liver and kidney. In addition, Me2GlyDH mRNA could be demonstrated in total RNA samples of lung, heart and brain but was barely detectable in spleen total RNA. In RNA prepared from 14-day rat embryos, Me2GlyDH-specific mRNA was clearly present. Among various liver cell types, besides hepatocytes, endothelial cells showed a high level of Me2GlyDH mRNA expression. There was no amplification product detectable in liver macrophages (Kupffer cells) and only a very faint one in fat-storing cells (Ito cells). Western blots confirmed at the protein level the predominant expression of the enzyme in liver and kidney, but Me2GlyDH protein was also present in the protein extract of lung, heart, spleen and brain. Immunohistochemical staining of liver slices with Me2GlyDH-specific antiserum revealed that expression of this enzyme is evenly distributed throughout the liver tissue. In the kidney, expression of the enzyme was located in the proximal tubule cells. Our results demonstrate that, contrary to the previously assumed liver-restricted expression, this enzyme is specifically expressed predominantly in the liver and kidney, but, in addition, it is detectable in many other tissues of the rat.

Aging↗
PubMed2005

Novel multiple 5'-amino-modified primer for DNA microarrays.

For DNA microarray analysis, total RNA is reverse-transcribed, labeled by incorporating fluorescent dye into the cDNA, and used to hybridize microarray. This protocol requires a minimum of 20 microg of total RNA. To overcome the sample limitation, an RNA amplification technique has been developed. Although it needs less RNA, this amplification technique is relatively expensive, time consuming, and, unfortunately, has been found to introduce bias. In this study, we designed a novel 5'-amino-modified primer and used it for priming cDNA synthesis. The novel primer has a special structure that contains four Uni-Link molecules with two nucleotide (thymine) residues inserted between them as spacers. This novel primer is used in the reverse-transcription reaction for cDNA synthesis. Using the novel 5'-modified primer combined with indirect labeling method, cDNA probes can be prepared with much less total RNA (5 microg or less) without amplification producing optimal results after hybridization of arrays. This primer can also be used to label nucleotides for other purposes.

Cell Line, Tumor↗
PubMed1979

RNA synthesis in permeable mouse ascites sarcoma cells.

A permeable cell system for studying RNA synthesis was established. Mouse ascites sarcoma cells were made permeable to nucleoside triphosphates and alpha-amanitin by treating with a hypotonic buffer. Separate determinations of endogenous RNA polymerase I, II and III activities in permeable cells were conducted using the different sensitivities of these enzymes to alpha-amanitin. The endogenous activity of RNA polymerase II under optimal conditions was one tenth of total RNA synthetic activity in isolated nuclei, and one third of that in permeable cells. The extremely low ratio of RNA polymerase II activity to total RNA synthetic activity in isolated nuclei was thought to be caused by increase of RNA polymerase I activity and decrease of RNA polymerase II activity. These and other results suggested that RNA synthesis in permeable cells reflects more precisely the in vivo state of RNA synthesis than thatin isolated nuclei. The permeable cell system will provide a useful method for studying the separate activities of RNA polymerases I, II and III in situ.

Amanitins↗
PubMed1993

Ovarian thecal/interstitial androgen synthesis is enhanced by a follicle-stimulating hormone-stimulated paracrine mechanism.

To obtain direct evidence for FSH-stimulated paracrine signaling in the ovary, 21-day-old intact or hypophysectomized female Wistar rats received four sc injections of recombinant human FSH (rhFSH; total dose, 16-72 IU) at 12-h intervals. Ovaries were removed 48 h after the first injection to extract total RNA for Northern analysis of 17-hydroxylase/C17-20-lyase (cytochrome P450c17 alpha) mRNA or to isolate thecal/interstitial cells for assessment of basal and hLH-responsive androgen synthesis in vitro. In situ hybridization with a 35S-labeled cytochrome P450c17 alpha cRNA probe confirmed that expression of the cytochrome P450c17 alpha gene was specific to thecal/interstitial cells. The approximately 2.0-kilobase P450c17 alpha mRNA signal in ovarian total RNA from intact animals was increased approximately 5-fold by treatment with rhFSH (total dose, 72 IU) or PMSG (15 IU). This effect was shown to be dose dependent, with a approximately 2-fold increase in response to 16 IU (total dose) rhFSH. P450c17 alpha mRNA levels in isolated granulosa and thecal/interstitial cell total RNA from intact animals were compared to establish which was the principal cellular site of P450c17 alpha mRNA expression. The P450c17 alpha mRNA signal was undetectable in control granulosa cells and only barely discernible after treatment with 72 IU (total dose) rhFSH. In contrast, P450c17 alpha mRNA was abundant in control thecal/interstitial mRNA, and its level was increased 4- to 6-fold by treatment with rhFSH. Treatment of hypophysectomized animals with rhFSH did not consistently alter ovarian P450c17 alpha mRNA levels. During culture for 48 h in serum-free medium, basal androgen (androstenedione plus androsterone) production by thecal/interstitial cells from intact animals was unaffected by treatment with rhFSH in vivo, but hLH-stimulated androgen production by these cells was enhanced approximately 2-fold. Neither basal nor hLH-responsive androgen production by thecal/interstitial cells from hypophysectomized animals was altered by previous treatment with rhFSH in vivo. Treatment of thecal/interstitial cell cultures from both intact and hypophysectomized animals with inhibin (0.1-30 ng/ml), a putative granulosa-derived paracrine factor, did not measurably affect basal androgen synthesis, but potently enhanced LH-responsive androgen synthesis in vitro. Similarly, treatment of thecal/interstitial cell cultures with conditioned medium from FSH-treated granulosa cell cultures significantly enhanced LH-responsive, but not basal, androgen production. We conclude that treatment of pituitary-intact rats with "pure" FSH modulates thecal/interstitial cell androgen synthesis. Granulosa cells, but not thecal cells, possess FSH receptors, and thecal/interstitial cells are the principal ovarian sites of P450c17 alpha expression.(ABSTRACT TRUNCATED AT 400 WORDS)

Androgens↗
PubMed1992

High sensitivity quantification of RNA from gels and autoradiograms with affordable optical scanning.

To increase sensitivity and to improve normalization of RNA levels in Northern blot analysis, a comparatively inexpensive optical scanner was utilized for digitizing photonegatives of ethidium bromide stained gels and autoradiograms. The optical scanner captures the image with a maximum resolution of 300 dots per inch by assigning one of 256 gray levels (8-bit) to each dot in the image. With the use of the public domain NIH Image program (requires a Macintosh II and an 8-bit video card), gel or autoradiogram bands in the digitized image are selected and their average gray scale density measured. We found that the digitized image of a photonegative of a TAE (Tris-acetate/EDTA) agarose gel, loaded incrementally with 50-1500 ng total RNA, produced a linear response over a 4-fold range down to 100 ng (R2 greater than 0.950). In utilizing "quantification" gels like this, RNA samples that are too dilute or too small for traditional spectrophotometric techniques can be normalized and loaded uniformly onto subsequent Northern gels. Results from autoradiogram scans demonstrate highly linear gray scale responses over a 4-fold range of total RNA (R2 greater than 0.950) that are reproducible with different blots and probe types (e.g., riboprobe, cDNA and oligonucleotide). In addition, we describe a normalization technique using a 30-mer oligonucleotide probe for rat 28S ribosomal RNA as a measure of total RNA loaded per gel lane. Altogether, this scanning, ribosomal RNA normalization system allows the measurement of relative changes between 20% and 400% using standard autoradiographic methods.

Actins↗
PubMed2004

A comparative analysis of data generated using two different target preparation methods for hybridization to high-density oligonucleotide microarrays.

BACKGROUND: To generate specific transcript profiles, one must isolate homogenous cell populations using techniques that often yield small amounts of RNA, requiring researchers to employ RNA amplification methods. The data generated by using these methods must be extensively evaluated to determine any technique dependent distortion of the expression profiles. RESULTS: High-density oligonucleotide microarrays were used to perform experiments for comparing data generated by using two protocols, an in vitro transcription (IVT) protocol that requires 5 microg of total RNA and a double in vitro transcription (dIVT) protocol that requires 200 ng of total RNA for target preparation from RNA samples extracted from a normal and a cancer cell line. In both cell lines, about 10% more genes were detected with IVT than with dIVT. Genes were filtered to exclude those that were undetected on all arrays. Hierarchical clustering using the 9,482 genes that passed the filter showed that the variation attributable to biological differences between samples was greater than that introduced by differences in the protocols. We analyzed the behavior of these genes separately for each protocol by using a statistical model to estimate the posterior probability of various levels of fold change. At each level, more differentially expressed genes were detected with IVT than with dIVT. When we checked for genes that had a posterior probability greater than 99% of fold change greater than 2, in data generated by IVT but not dIVT, more than 60% of these genes had posterior probabilities greater than 90% in data generated by dIVT. Both protocols identified the same functional gene categories to be differentially expressed. Differential expression of selected genes was confirmed using quantitative real-time PCR. CONCLUSION: Using nanogram quantities on total RNA, the usage of dIVT protocol identified differentially expressed genes and functional categories consistent with those detected by the IVT protocol. There was a loss in sensitivity of about 10% when detecting differentially expressed genes using the dIVT protocol. However, the lower amount of RNA required for this protocol, as compared to the IVT protocol, renders this methodology a highly desirable one for biological systems where sample amounts are limiting.

Algorithms↗
PubMedDate not supplied

Isolation of poly(A)-rich mRNA from the chorioallantoic membrane of Sendai virus-infected chick embryos.

Total RNA was extracted from the chorioallantoic membrane (CAM) of Sendai virus-infected chick embryos by the original Kecskeméthy-Schäfer method and by a modification of this technique, including phenol treatment, in order to ensure complete removal of proteins. The purity of the total RNA extracted by the modified technique was ascertained by spectral analysis. Chromatography on oligo(dT)-cellulose of the total RNA led to the isolation of poly(A)-rich mRNA--the material retained by oligo(dT)-cellulose and eluted with the buffer of the lowest ionic strength. The ability of the poly(A)-rich mRNA thus obtained to stimulate incorporation of 14C-leucine into protein was demonstrated in a cell-free protein-synthesizing assay system.

Allantois↗
PubMed1976

Thermosensitivity of nuclear RNA polymerase during the in vitro senescence of mouse fibroblasts.

Embryonic mouse fibroblasts divide approximately twelve times in vitro prior to cessation of mitotic activity. During this period of cellular senescence the thermosensitivity of the RNA polymerase activity of isolated nuclei has been examined as a means of detecting the possible accumulation of defective enzyme molecules, as has been found by other workers for several cytoplasmic enzymes during the ageing of human fibroblasts in vitro. The total RNA polymerase activity of nuclei isolated from old (10th generation) cells is more thermoresistant than that of young (2nd generation) cells. However, the net RNA polymerase activity of nuclei from non-dividing (confluent) cells is more thermoresistant than that of exponentially growing cells of the same age. When allowance is made for the state of growth of the cultures, little difference is seens in the thermosensitivity of the activities of nuclei from old and young cells. Neither is there any difference between the thermosensitivity of the net activity of an established line of murine fibroblasts (L-cells) and cells in primary culture. Preheating nuclei increases the inhibition of their total RNA polymerase activity by alpha-amanitin, indicating that RNA polymerase II is the most heat resistance species present. There appears to be no difference between the thermosensitivity of the alpha-amanitin sensitive and resistance species of the enzyme in the nuclei of old and young cells. It is concluded that old cells resemble non-dividing young cells in containing a higher proportion of RNA polymerase II in their nuclei, resulting in greater thermoresistance of the total RNA polymerase activity over that of exponentially growing cells. However, there appears to be no increase in thermosensitivity of the enzymes with age.

Amanitins↗
PubMed1984

Myosin heavy chain mRNA during the development and regression of myocardial hypertrophy.

RNA synthesis increases during the development and decreases during the regression of pressure-overload induced myocardial hypertrophy. Until now, we have been unable to determine whether these events actually reflected changes in the messenger RNAs for the myosin heavy chain mRNA in the total RNA extracted from rat heart during the development and regression of hypertrophy. We found that the amount of MHC mRNA increased in proportion to the overall increase in total RNA during the development of hypertrophy. However, upon relief of pressure-overload, MHC mRNA decreased to a much greater degree than did total RNA. This is probably related to a sudden cessation of synthesis and the rapid degradation of mRNA. We conclude that the development and regression of myocardial hypertrophy is a function of changes in the mRNA for contractile proteins.

Animals↗