Total Cellular mRNA Research Articles

Acquisition of embryogenic potential in carrot cell-suspension cultures

Embryogenic suspension cultures of domesticated carrot (Daucus carota L.) are characterized by the presence of proembryogenic masses (PEMs) from which somatic embryos develop under conditions of low cell density in the absence of phytohormones. A culture system, referred to as starting cultures, was developed that allowed analysis of the emergence of PEMs in newly initiated hypocotyl-derived suspension cultures. Embryogenic potential, reflected by the number of FEMs present, slowly increased in starting cultures over a period of six weeks. Addition of excreted, high-molecular-weight, heat-labile cell factors from an established embryogenic culture considerably accelerated the acquisition of embryogenic potential in starting cultures. Analysis of [(35)S]methionine-labeled proteins excreted into the medium revealed distinct changes concomitant with the acquisition of embryogenic potential in these cultures. Analysis of the pattern of gene expression by in-vitro translation of total cellular mRNA from starting cultures with different embryogenic potential and subsequent separation of the [(35)S]methionine-labeled products by two-dimensional polyacrylamide gel electrophoresis demonstrated a small number of abundant in-vitro-translation products to be present in somatic embryos and in embryogenic cells but absent in nonembryogenic cells. Several other in-vitro-translation products were present in explants, non-embryogenic and embryogenic cells but were absent in somatic embryos. Hybridization of an embryoregulated complementary-DNA sequence, Dc3, to RNA extracted from starting cultures showed that the corresponding gene is expressed in somatic embryos and PEMs but not in non-embryogenic cells.

Increased Elastin Production by Progeria Skin Fibroblasts is Controlled by the Steady-State Levels of Elastin mRNA

Hutchinson-Gilford progeria is a unique, rare disease with markedly accelerated aging. The average lifespan of affected individuals is 12 years. Although the biochemical basis of the syndrome is unknown, its influence appears to be primarily upon mesodermal tissues. Characteristics such as the altered appearance of the skin and the extensive and fatal involvement of the cardiovascular system led us to study elastin production in cultured skin fibroblasts from three progeroid individuals. We found tropoelastin production by progeroid cells was elevated six- to nine-fold at the protein and mRNA levels, while relative collagen synthesis was similar to control strains. There was little difference between progeroid and normal cells in expression of total protein or in total cellular mRNA content. Western blot analysis of tropoelastin from progeroid fibroblasts confirmed increased production of elastin but revealed no gross changes in the molecular mass. The significant increase in tropoelastin expression lends support to the concept that progeria results from a mesenchymal dysplasia, and offers a possible biochemical marker for the phenotype.

A high-efficiency vector for expression of foreign genes in myeloma cells.

We have constructed a high-efficiency vector for expression of genes of interest in myeloma cells. This vector is comprised of regulatory sequences from immunoglobulin (Ig) genes, including the heavy-chain enhancer, a kappa light-chain promoter and splice site, and the polyadenylation signal downstream from the kappa constant region. The expression capacity of this vector was assayed in J558L myeloma cells using human tissue plasminogen activator as a reporter gene. Stable transfectants were analyzed for protein, RNA, DNA copy number and transcription rate. Expression was compared to that of intact, transfected Ig genes and to endogenous Ig. Tissue plasminogen activator cloned into this vector was found to be expressed as efficiently as intact, transfected Ig genes, producing 1-2% of total cellular mRNA from a single copy of the gene. RNA levels and transcription rates relative to those of endogenous Ig genes were found to be about 25% and 38%, respectively. Because of its high efficiency, potential for gene amplification, and various scale-up advantages of myeloma cells, this vector-host system may yield levels of desired proteins than currently available systems.

Rapid induction by fungal elicitor of the synthesis of cinnamyl-alcohol dehydrogenase, a specific enzyme of lignin synthesis.

A fivefold increase in the extractable activity of cinnamyl-alcohol dehydrogenase, an enzyme of phenylpropanoid metabolism specific for lignin synthesis, was observed within 10 h of treatment of cell-suspension cultures of bean (Phaseolus vulgaris L.) with a high-molecular-mass elicitor preparation heat-released from mycelial cell walls of the bean pathogen Colletotrichum lindemuthianum. Elicitor caused a rapid, marked but transient increase in the synthesis of cinnamyl-alcohol dehydrogenase with maximum rates 2-3 h after elicitation, concomitant with the phase of rapid increase in enzyme activity. There is a close correspondence between increased polysomal mRNA activity encoding cinnamyl-alcohol dehydrogenase, as measured by incorporation of [35S]methionine into immunoprecipitable enzyme subunits in vitro, and the stimulation of enzyme synthesis in vivo in response to elicitor. This marked increase in polysomal mRNA activity represents an increase as a proportion of total cellular mRNA activity, indicating that elicitor does not stimulate synthesis of this enzyme by selective recruitment from the total pool of cellular mRNA. Elicitor stimulation of cinnamyl-alcohol dehydrogenase activity and enzyme synthesis is more rapid than previously observed for other proteins involved inducible defense mechanisms, such as enzymes of phytoalexin biosynthesis or the apoproteins of cell-wall hydroxyproline-rich glycoproteins.

Transcription of multiple copies of the yeast GAL7 gene is limited by specific factors in addition to GAL4.

High levels of the GAL7 gene in the yeast cell appear to titrate regulatory factors and to impair transcription of related sequences. To investigate the role that the GAL regulatory factors GAL4 and GAL80 have in this process we have compared the accumulation of mRNA transcribed from single-copy (plasmid-borne GAL7 and chromosomal GAL10) and high-copy (plasmid-borne GAL7) genes in several GAL regulatory mutants. Our results show that functional GAL4 gene product is required for induction of transcription from the single- and high-copy genes. In a strain containing the GAL4 gene fused to the high expression ADH1 promoter, glucose can replace galactose to induce high levels of transcription of GAL7 and GAL10 genes, although the kinetics of accumulation induced by the two sugars are distinctly different. In the presence of high levels of GAL4, maximum accumulation of mRNA from single and high copy genes is elevated two-fold; disruption of the gal80 gene in combination with high levels of GAL4 results in a further two-fold increase in transcription. In this genetic background, galactose-induced transcription of the high copy GAL7 gene results in a greater than 50-fold increase in the levels of GAL7 mRNA, representing 30%-50% of the total cellular mRNA. Our results are consistent with a cooperative effect of saturation of multiple GAL4 DNA binding sites and with a limiting factor, in addition to GAL4, that is required for transcription of the GAL genes.

Effects of starvation on rat liver mRNA translation products.

The synthesis of rat liver protein and RNA decreases with starvation. It is not yet known whether such decreases are regulated strictly at a transcriptional level, or if post-transcriptional controls are also involved. In this study we investigate the effects of 0, 2, or 4 days starvation on the levels of specific, abundant mRNAs in total and polysomal RNA populations. The mRNAs were analyzed by translation in vitro in mRNA-dependent, cell-free, protein synthesizing systems. The resulting polypeptide products were separated by gel electrophoresis and visualized with fluorography. The amount of albumin translated from both polysomal and total cellular mRNA decreased 20-40% with fasting. In contrast, a specific peptide having a molecular mass of approximately 30 kDa increased two- to three-fold in total cellular RNA with a smaller increase observed in polysomal RNA. These changes were maximal at 2 days of starvation. Since starvation is known to cause alterations in liver metabolism the 30-kDa polypeptide may be related to enzymes or other proteins involved in this homeostatic response.

Effects of insulin alone on the accumulation of a specific mRNA in rat hepatoma cells.

Experiments were conducted to investigate the action of insulin alone on the induction of a specific mRNA referred to as p33. In 24-h serum-deprived rat liver hepatoma cells (H4) grown in monolayer cultures, insulin induced a 3-fold increase in the levels of p33 mRNA within 30 min, and a maximum 10-fold increase was observed by 1 h. The effects of insulin were evident at concentrations as low as 10(-12) M and were maximal at 0.5-1 X 10(-8) M. The effect of insulin was specific, since the level of mRNA for the histocompatibility complex, H2d, was unaffected by insulin. The increase in p33 mRNA was not due to an effect of insulin on the stabilization of mRNA, since insulin did not alter the half-life of this message. In addition, two different RNA synthesis inhibitors blocked the stimulation of p33 mRNA production by insulin. These data suggest that insulin may specifically stimulate the synthesis of p33 mRNA which results in an increased accumulation of total cellular p33 mRNA.

Differential post-translational modification of human type I keratins synthesized in a rabbit reticulocyte cell-free system

The translation products synthesized in a rabbit reticulocyte lysate system, from total cellular mRNA from the human epithelial cell-line ME-180, have been examined. Keratin proteins are prominent among these translation products, and they precisely coelectrophorese in sodium dodecyl sulfate-polyacrylamide gels with keratins purified from the cells. Type-I, acidic, keratins which are acetylated in vivo, are also acetylated by the reticulocyte lysate. Examination by two-dimensional electrophoresis, of two acidic keratins known to be phosphorylated in vivo reveals that only one of these proteins is phosphorylated in the lysate system. Phosphorylation of this protein occurs after release of the completed polypeptide chain from the ribosome. The protein phosphorylated by the lysate is known to be the only ME-180 phosphokeratin modulated by cyclic AMP, reflecting in vitro the differential modification of ME-180 keratins in vivo.

Rapid regulation of L-type pyruvate kinase mRNA by fructose in diabetic rat liver.

The effect of fructose on the induction of L-type pyruvate kinase mRNA in diabetic rat liver was studied by using a cloned cDNA probe. Fructose feeding resulted in a 5- to 6-fold increase in the L-type enzyme mRNA level after 1 to 3 days. These changes were approximately proportional to the changes in the level of translatable mRNA of this enzyme. A significant increase in total cellular L-type enzyme mRNA level was observed within 2 h after fructose feeding and the level reached a maximum after 8 h. Dietary glycerol also markedly increased the L-type mRNA level. These alterations were essentially due to the changes in the cytosolic mRNA. Northern blot analysis of total cellular RNA revealed that two L-type enzyme mRNA species with molecular sizes of 2.1 and 3.6 kilobases were proportionally increased during the fructose induction. The two mRNA forms were found in immunopurified L-type enzyme mRNA and directed synthesis of the L-type subunit in vitro; they are therefore functional mature forms. In contrast, analysis of nuclear RNA showed five putative precursor RNA species for the enzyme, up to 9.4 kilobases in length, in the liver of fructose-fed rats, while no band of the RNA species was found in the nuclei of control liver. The changes in the number of bands of these RNA species and their intensities after fructose feeding preceded the changes in the level of total cellular L-type enzyme mRNA sequences. These results indicate that dietary fructose causes a rapid increase in the level of L-type pyruvate kinase mRNA sequences by acting at the nuclear level.

Evidence for the synthesis of multiple pro-opiomelanocortin-like precursors in murine leydig tumor cells

The opiate peptide β-endorphin has recently been localized in extrapituitary tissues and cells, including the Leydig cells of the testis, by immunohistochemical techniques. An intriguing question is whether this localization reflects an accumulation of the peptide through a specific uptake mechanism or is the result of synthesis within the cell in a large precursor form similar to pro-opiomelanocortin synthesized in the pituitary. Evidence is presented herein that specific antibodies against β-endorphin and adrenocorticotrophic hormone precipitate identical precursor molecules from total cellular mRNA translation products of M5480A Leydig tumor cells. In addition, mRNA from these cells cross-hybridizes under stringent conditions with a cDNA coding for the rat pituitary pro-opiomelanocortin sequence. These data demonstrate the synthesis of a pro-opiomelanocortin-like mRNA in this Leydig cell tumor and strongly implicate biosynthesis within these cells.

Induction of phenylalanine ammonia-lyase and 4-coumarate:CoA ligase mRNAs in cultured plant cells by UV light or fungal elicitor

The mRNAs encoding two enzymes of phenylpropanoid metabolism, phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) and 4-coumarate:CoA ligase (4CL; EC 6.2.1.12), were induced in cultured parsley cells (Petroselinum hortense) either by irradiation with UV light or by treatment with elicitor, a cell-wall fraction of the fungus Phytophthora megasperma f. sp. glycinea. Two-dimensional gel electrophoresis of the encoded PAL and 4CL proteins revealed that the mRNAs induced by either treatment were very similar if not identical. RNA blot hybridization with cDNAs complementary to these mRNAs was used to measure changes in the mRNA amounts at various times after either treatment. Total cellular PAL and 4CL mRNA amounts increased coordinately after UV irradiation to a maximum at 7 hr and then decreased to uninduced levels by 30 hr with the same kinetics as observed previously for the changes in the translational activities. Treatment with the fungal elicitor also caused coordinated, but more rapid, changes in PAL and 4CL mRNA translational activities, with a sharp peak occurring 3 hr after the addition of elicitor. Corresponding changes in mRNA amounts were observed only for 4CL, whereas the amount of PAL mRNA continued to increase at least up to 20 hr after elicitor addition. Our results suggest that parsley cells respond to UV irradiation or addition of fungal elicitor by increased rates of transcription of genes involved in the synthesis of compounds related to UV or disease resistance, respectively.

Absence of functional RNA encoded by a silent chromosome in non-complementing diploids obtained from protoplast fusion in Bacillus subtilis.

The molecular basis for lack of phenotypic expression of one chromosome in Bacillus subtilis non-complementing diploid clones (Ncd cells) was investigated. Correlations between chromosomal inactivation and absence of functional transcripts were determined with wild-type prophage phi 105 or a thermoinducible mutant phi 105 cts23, on either the expressed or the silent chromosome. It appears that no significant amount of phage mRNA is detectable in Ncd cells carrying the prophage in the inactive chromosome. However, phi 105 mRNA represents 0.23% of total cellular mRNA in an Ncd strain with the prophage in the expressed chromosome and 0.28% in the parental lysogenic strain. The lack of an mRNA repressor of phi 105 prophage from the silent chromosome was confirmed by the absence of repressor activity in Ncd clones with a temperature sensitive mutant phi 105 located in the silent chromosome. After heat induction, no phage production was observed. As expected these clones do not exhibit phi 105 immunity when superinfected with the same phage. The combined data of the present and previous work suggest that control of phenotypic suppression of Ncd strains should, at the transcription level, involve a different DNA tertiary organisation in one of the two chromosomes.

Elicitor Induction of mRNA Activity

Changes in the activity levels of mRNAs encoding phenylalanine ammonia-lyase and chalcone synthase, two characteristic enzymes of phenylpropanoid biosynthesis, in elicitor-treated cells of dwarf French bean (Phaseolus vulgaris L.) have been investigated by immunoprecipitation of [35S]methionine-labelled enzyme subunits synthesised in vitro in an mRNA-dependent rabbit reticulocyte lysate translation system. Elicitor heat-released from cell walls of Colletotrichum lindemuthianum, the causal agent of anthracnose disease of bean, causes marked rapid increases in the polysomal activities of the mRNAs encoding the two enzymes concomitant with the phase of rapid increase in enzyme activity at the onset of phaseollin accumulation during the phytoalexin defence response. Increased polysomal mRNA activities encoding the two enzymes can be observed 30 min after elicitor treatment. The patterns of induction of the mRNA activities are broadly similar with respect to time and elicitor concentration although small but distinct differences between the enzymes were observed in the elicitor concentration giving maximum induction. There is a close correlation between the induction of polysomal mRNA activity and the induction of enzyme synthesis in vivo by elicitor treatment with respect to both the kinetics of induction and the dependence on elicitor concentration. The data indicate that elicitor stimulation of phenylalanine ammonia-lyase and chalcone synthase synthesis in vivo is largely a result of increased polysomal activity of the mRNAs encoding these enzymes. Similar patterns of induction of polysomal mRNA activity are observed with elicitor preparations from a variety of sources. The marked increases in polysomal mRNA activities encoding phenylalanine ammonia-lyase and chalcone synthase are increases as a proportion of total cellular mRNA activity, indicating that elicitor does not increase these polysomal mRNA activities by stimulation of selective recruitment from the total pool of cellular mRNA.

Development of the adrenergic phenotype: increase in adrenal messenger RNA coding for phenylethanolamine-N-methyltransferase.

Mechanisms regulating the developmental increase in the activity of adrenal phenylethanolamine-N-methyltransferase (PNMTase), an index of the adrenergic phenotype, were examined. Immunotitration indicated that the increase in catalytic activity in rat adrenal from birth to adulthood was attributable to increased numbers of PNMTase molecules, not enzyme activation. To determine whether the ontogenetic increase in PNMTase protein was associated with elevation of mRNA coding for PNMTase, cell-free translation was performed on total cellular mRNA obtained from adrenals at different ages. Translation in wheat-germ and reticulocyte lysate systems, followed by immunoprecipitation of the PNMTase product, NaDodSO4 gel electrophoresis, and fluorography, showed an 8-fold increase in the proportion of specific PNMTase mRNA relative to total mRNA in rat adrenals from birth to adulthood. Moreover, bovine adrenal medullae exhibited a 100-fold increase in PNMTase mRNA levels between embryonic life and adulthood. Consequently, the ontogenetic increase in adrenal PNMTase appears to be due to a developmental rise in specific mRNA coding for the protein.

Differential stability of α- and β-globin mRNAs after infection with herpes simplex virus

Friend erytholeukemia (FL) cells can be induced to undergo erythroid differentiation following. exposure to dimethyl sulfoxide (Me$JO) [l] or hemin [2] . The differentiation process involves a series of events which include, production of globin mRNA [3-51 expression of erythrocyte membrane antigens [6] and a decrease in the ability of induced cells to divide [7] . In cells exposed to either Me$O or hemin, both crand &globin mRNAs are synthesized [2,5]. It has been shown that the relative rate of accumulation of crand /.I-globin mRNAs differs with different inducing agents [8] . We have utilized this system to examine the fate of cellular mRNAs during the course of lytic infection with herpes simplex virus (HSV). We demonstrated that the de novo synthesis of globin was rapidly shut off early after infection with HSV and that the abundance of globin mRNA sequences decreased as the infection proceeded [9]. In a subsequent analysis of total infected cell mRNA we provided evidence which suggested that total polyadenylated cellular mRNA was degraded at 4 h postinfection and that the degradation of globin mRNA was a stochastic process as judged by the failure to detect a shift in the kinetics of hybridization of total polyadenylated mRNA extracted at various times postinfection to globin cDNA [IO] . To further explore these findings, specific cDNA probes to crand fi-globin mRNA were prepared and used to analyze the relative stability of crand /I-globin mRNAs following infection with HSV. The results demonstrate that degradation of cr-globin mRNA is more.refractile to HSV infection than

Studies on the stability of ribosomal protein messenger RNA

The functional stability of the messenger RNA for ribosomal proteins has been investigated by radioimmunoprecipitation with antisera prepared against ribosomal proteins. Ribosomal protein mRNA has been shown to decay with a half-life comparable to total cellular mRNA. The possible significance of these results has been discussed.

Use of Sepharose 4B for preparative scale fractionation of eukaryotic messenger RNA's.

Abstract A new technique using Sepharose 4B column chromatography for the partial purification of the total messenger RNA population of several animal tissues is described. The column when eluted with 0.1M sodium acetate, pH 5.0, containing. 001M EDTA, resolves a total nucleic acid extract into four major peaks. DNA is eluted at the void volume, followed successively by peaks of 18S ribosomal RNA, 4S transfer RNA and 28S ribosomal RNA. Ribonucleic acid containing mRNA activities is eluted after the DNA peak but immediately before the 18S rRNA peak. Hence the column enables quantitative removal of DNA, 5S RNA, tRNA and 28S rRNA from the majority of total cellular mRNA's. Partial segregation of mRNA's in the column is also demonstrated. The method does not require the isolation of polysomes as the initial procedure in mRNA isolation and is readily adaptable to large scale preparations. One hundred mg of total nucleic acid extracted from whole tissues can be fractionated on a 5 × 100 cm Sepharose 4B column. ...

Messenger RNA in HeLa cells: Kinetics of formation and decay

The polyadenylic acid-containing messenger RNA fraction of HeLa cells was measured by its affinity for oligedeoxythymidylate cellulose. Both the kinetics of initial labeling and the decay after a brief pulse of incorporation were examined. The kinetics of decay are complex, but can be approximated by assuming two populations; a short-lived species with a half-life of seven hours and a long-lived component with a half-life of 24 hours. It is estimated that the short-lived material comprises 33% of total cellular mRNA, while the relatively stable species amounts to 67% of the steady-state mRNA content. The two mRNA components with different decay times were observed simultaneously in the same cell population by measuring decay of 24-hour old mRNA labeled with 14C and RNA briefly labeled with 3H. The old mRNA had only a 24-hour decay component, while the new mRNA was biphasic. The decay of old and new mRNA was also observed after RNA synthesis was inhibited with actinomycin. Again, old mRNA decayed more slowly than recently labeled material. However, both decay times are significantly shorter in the presence of actinomycin and correspond to half-lives of approximately 4 and 12 hours. There is a small but significant difference in sedimentation distribution of new and old mRNA, the old mRNA sedimenting more slowly than new material, suggesting that the more stable species has a lower average molecular weight. The steady-state content of mRNA in HeLa cells amounts to 5.5% of the ribosomal RNA, or more than twice the amount of messenger RNA estimated to be on hemoglobin-synthesizing polyribosomes.