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At least 307 records · Page 17Linked to original sources
PubMed1984

Phosphorylation of tyrosine in cultured human placenta.

The [32P]phosphoamino acids in proteins of first-trimester and term-cultured human placentas have been separated and their relative amounts have been measured. Significant phosphorylation of tyrosine residues could be detected in the cultured placental tissue at different stages of gestation. The phosphotyrosine accounts for 2-4% of the total acid-stable phosphate in the phosphoamino acids after partial acid hydrolysis. The difference in the extent of [32P]tyrosine in various placentas seems to be a function of biological variation of the individual placentas, rather than a function of placental age and stage of gestation. In contrast, a significant difference in the phosphorylation ratio of serine and threonine could be measured between first-trimester and term placentas. As more evidence is accumulating that protein phosphorylation of tyrosine is involved in the processes of cellular growth and proliferation, our findings of the relatively high tyrosine phosphorylation in human placenta strongly suggest that this type of protein phosphorylation may play an important role in the placental growth and development. Furthermore, these findings may correlate with the existence of the endogenous RNA virus-like particles found in normal human placenta.

Culture Techniques↗
PubMed1983

Phosphorylation of tyrosine in cultured human placenta.

The [32P]phosphoamino acids in proteins of first trimester and term-cultured human placentas have been separated and their relative amounts were measured. A significant phosphorylation of tyrosine residues could be detected in the cultured placental tissue at different stages of gestation. The phosphotyrosine accounts for 2-4% of the total acid-stable phosphate in the phosphoamino acids after partial acid hydrolysis. The difference in the extent of [32P]tyrosine in various placentas seems to be a function of biological variation of the individual placentas, rather than a function of placental age and stage of gestation. In contrast, a significant difference in the phosphorylation ratio of serine and threonine could be measured between first trimester and term placentas. As more evidence is accumulating that protein phosphorylation of tyrosine is involved in the processes of cellular growth and proliferation, our findings of the relatively high tyrosine phosphorylation in human placenta strongly suggest that this type of protein phosphorylation may play an important role in the placental growth and development. Furthermore, these findings may correlate with the existence of the endogenous RNA virus-like particles found in normal human placenta.

Female↗
PubMed1984

Response of isolated nuclei to phospholipid vesicles: analysis of the nuclear proteins after treatment with phosphatidylserine and phosphatidylcholine and comparison with heparin.

Phospholipid liposomes affect the histone pattern of isolated rat liver nuclei. Multilamellar vesicles (MLV) obtained with phosphatidylserine (PS) release a large amount of the lysine rich histones, while those obtained with phosphatidylcholine (PC) do not induce significant changes with respect to controls. This different response has been compared to the effects obtained with Heparin, which slightly modifies the relative ratio of the histone fractions. These data might account for the mode by which phospholipids induce transitions of the chromatin structure and changes of the endogenous RNA polymerase activity.

Animals↗
PubMed2004

Cell-free protein synthesis using cell extract of Pseudomonas fluorescens and CspA promoter.

We have modified the cell-free coupled transcription/translation system of bacteria. The cell-free extract of Pseudomonas fluorescens was used for translation instead of Escherichia coli. In addition, transcription of the target gene was regulated by CspA promoter with endogenous RNA polymerase instead of by T7 promoter with exogenous T7 RNA polymerase. We could increase the yields of soluble proteins using different combinations of the S30 extract and the promoter and different temperatures for protein synthesis. Increasing the variety of synthesis systems allows production of large quantities of soluble proteins. In order to carry out efficient cell-free protein synthesis, versatile pCop-plasmids carrying CspA promoter were constructed and these plasmids were applicable to expression of recombinant proteins in E. coli cells.

Amino Acid Sequence↗
PubMed2026

Deciphering differential mRNA and lncRNA expression profiles in response to PEG simulated drought stress in cucumber (Cucumis sativus L.).

Cucumber (Cucumis sativus L.), a vital fruit vegetable of the Cucurbitaceae family, originated in India ∼ 3000 years ago. It is widely used in the culinary, therapeutic, and cosmetic sectors. Cucumber cultivation is significantly impacted by drought stress, especially in arid and semi-arid regions. This study investigates the molecular response to drought using two contrasting cucumber lines: WBC-23-2 (drought-tolerant) and DGPC-59 (drought-sensitive). Drought was simulated using polyethylene glycol (PEG), and effects on physiological and biochemical traits were evaluated. The tolerant line exhibited reduced leaf wilting and higher relative water content (RWC). Based on these physiological markers, transcriptomic profiling was employed to identify the underlying regulatory networks. Analysis identified 4,736 DEGs, suggesting that the tolerant line's superior resilience is driven by preferential activation of genes involved in photosynthesis and glutathione metabolism. Conversely, the sensitive genotype showed enrichment in organonitrogen compound catabolism and water deprivation response. This divergence is further reflected in the regulation of 155 transcription factors (TFs) across various families, indicating distinct regulatory architectures between the two lines. Additionally, 774 drought-responsive long non-coding RNAs (lncRNAs) were identified, acting via cis, trans, and competing endogenous RNA (ceRNA) mechanisms to modulate gene expression. Key candidate genes associated with drought tolerance included WAT1-related protein At5g64700, thaumatin-like protein, berberine bridge enzyme-like 18, probable WRKY transcription factor, and pathogenesis-related protein 1. This study reveals a complex regulatory network of mRNAs, lncRNAs, and TFs underlying drought response and provides a valuable foundation for breeding drought-resilient cucumber cultivars. A web-based genomic resource, CsDTDb, has been developed and made publicly available to facilitate future functional genomics studies related to drought tolerance in cucumber.

DEGs↗
PubMed2005

Development of an inducible protein expression system based on the protozoan host Leishmania tarentolae.

Production of functional eukaryotic proteins in recombinant form is a bottle-neck in various post-genomic applications and in life science in general. At least partially this is due to the problems associated with the use of endogenous RNA polymerase II for high-level transcription of heterologous genes in eukaryotic expression systems. To circumvent these problems we developed a new inducible protein expression system based on the protozoan host Leishmania tarentolae (Trypanosomatidae). We have created a strain of L. tarentolae constitutively co-expressing T7 RNA polymerase and tetracycline repressor. This strain could be stably transformed with the heterologous target gene under control of the T7 promoter/TET operator assembly, which can initiate transcription upon addition of tetracycline to the culture medium. Using this system, we demonstrated that enhanced green fluorescent protein (EGFP) could be overexpressed to a level of ca. 1% of total cellular protein. The developed system was tested for its ability to inducibly co-express multiple genes. Using two copies of the egfp gene integrated at two different genomic sites, we could obtain expression levels reaching 4% of total cellular protein. Further possible improvements and applications of the developed system are discussed.

Animals↗
PubMed1998

Are tRNAs imported into the mitochondria of kinetoplastid protozoa as 5'-extended precursors?

All mitochondrial tRNAs in kinetoplastid protozoa are encoded in nuclear DNA and transported into the mitochondrion (Simpson et al., Nucl Acids Res 1989;17:5427-5445; Hancock and Hajduk, J Biol Chem 1990;265:19208-19215). It has been proposed that tRNAs in these cells are imported into the mitochondrion as 5'-extended precursors which are processed by a mitochondrial RNase P-like activity (Hancock et al., J Biol Chem 1992;267:23963-23971). We have examined this hypothesis by cloning and sequencing primer extension products of mitochondrial tRNAs from Leishmania tarentolae and Trypanosoma brucei, and have found that these are derived from circularized mature tRNA molecules. We suggest that these molecules are produced by the endogenous RNA ligase activity (Bakalara et al., J Biol Chem 1989;264:18679-18686) either in vivo or during mitochondrial isolation. We did not obtain any evidence for the existence of high molecular weight precursors of mitochondrial tRNAs. This negative result is consistent with previous in vivo transfection studies with both L. tarentolae (Lima and Simpson, RNA 1996;2:429-440) and T. brucei (Hauser and Schneider, EMBO J 1995;14:4212-4220; Schneider et al., Mol Cell Biol 1994;14:2317-2322), in which mitochondrial targeting of plasmid-expressed tRNAs was independent of the presence of 5'-flanking sequences. We conclude that the hypothesis for 5'-extended tRNA precursors in kinetoplastid mitochondrial importation remains to be verified.

Animals↗
PubMed2003

Cytoplasmic polyhedrosis virus structure at 8 A by electron cryomicroscopy: structural basis of capsid stability and mRNA processing regulation.

The single-shelled cytoplasmic polyhedrosis virus (CPV) is a unique member of the Reoviridae. Despite lacking protective outer shells, it exhibits striking capsid stability and is capable of endogenous RNA transcription and processing. The 8 A three-dimensional structure of CPV by electron cryomicroscopy reveals secondary structure elements present in the capsid proteins CSP, LPP, and TP, which have alpha+beta folds. The extensive nonequivalent interactions between CSP and LPP, the unique CSP protrusion domain, and the perfect inter-CSP surface complementarities may account for the enhanced capsid stability. The slanted disposition of TP functional domains and the stacking of channel constrictions suggest an iris diaphragm-like mechanism for opening/closing capsid pores and turret channels in regulating the highly coordinated steps of mRNA transcription, processing, and release.

Animals↗
PubMed1978

Synthesis of mouse mammary tumor virus ribonucleic acid in isolated nuclei from cultured mammary tumor cells.

Glucocorticoid hormone treatment of GR cells, a cultured line derived from mouse mammary tumor tissue, selectively stimulates the rate of transcription of integrated proviral genes specifying mammary tumor virus (MTV). We have incubated isolated nuclei from these cells under conditions in which all three endogenous RNA polymerases appear to be active. RNA synthesized in vitro is distinguished from preexisting nuclear RNA by labeling the in vitro products with [3H]CTP, and the level of MTV RNA synthesis is measured by molecular hybridization with unlabeled viral DNA. Synthesis requires the addition of nucleoside triphosphates, and is inhibited by actinomycin D. Pretreatment of GR cells with dexamethasone, a synthetic glucocorticoid, has no significant effect on the amount of total RNA synthesis in isolated nuclei. In contrast, synthesis of MTR RNA is stimulated 10-20-fold in nuclei from dexamethasone-treated cells relative to untreated control nuclei; the sensitivity of in vitro viral RNA synthesis to inhibition by alpha-amanitin suggests that it is carried out exclusively by RNA polymerase II. The fraction of total RNA synthesis which is viral specific (about 0.2-0.4% in nuclei from dexamethasone-treated cells and 0.01-0.03% in controls) is similar to that detected in pulse labeled RNA from whole GR cells in culture. Our procedures for labeling and hybridization of RNA appear to avoid artifacts recently noted in other in vitro transcription systems.

Amanitins↗
PubMed1976

Poly(adenylic acid) synthesis in isolated rat liver mitochondria.

Purified rat liver mitochondria were shown to synthesize poly(adenylic acid) (poly(A)) in vitro. Detection of the poly(A) synthesizing activity was facilitated by addition of NaF to the reaction was shown to be poly(A) by its insensitivity to digestion with pancreatic RNase and RNase T1, its degradation by venom phosphodiesterase and its retention on poly (uridylic acid) 20-23 AMP units and it was covalently attached to the endogenous RNA in the mitochondria. Poly(A) synthesis required ATP and a divalent ion and was maximally active in the pH range of 7-8. The reaction was inhibited by atractyloside, cordycepin triphosphate, Rose Bengal, rifamycin derivative AF/103, sodium pyrophosphate, and N-ethylmaleimide. These studies indicate that the mitochondrial poly(A) polymerase previously described in our laboratory (Jacob, S.T., Rose, K.M., and Morris, H.P. (1974), Biochim. Biophys. Acta 361, 312-320) is involved in the posttranscriptional addition of poly(A) sequence to mitochondrial RNA.

Adenosine Triphosphate↗
PubMed2004

Sequence-specific detection of MicroRNAs by signal-amplifying ribozymes.

The rational and straightforward design of hairpin ribozymes that can be sequence-specifically induced by external oligonucleotides is described. Due to intrinsic signal amplification, their sensitivity is at least an order of magnitude increased compared to standard molecular beacons. We applied this system to the detection of microRNAs, a recently discovered class of small endogenous RNA molecules that are involved in gene regulation. We show that the cognate microRNA can reliably and sensitively be detected at low concentrations in a mix of other microRNA sequences. These probes may be useful in applications that require direct detection of minute amounts of small DNAs or RNAs.

Animals↗
PubMed1971

Protease and ribonuclease activities in bovine pituitary lobes.

1. Acid and alkaline protease activities in bovine anterior and posterior pituitary lobes were reinvestigated by measurement of u.v. and Folin-Ciocalteu colour values of trichloroacetic acid-soluble digestion products of denatured haemoglobin. 2. Both lobes of the pituitary gland contain a cathepsin with a pH optimum at 3.8. 3. When release of u.v.-absorbing material was used as the assay there was also an optimum at pH8.3-9.7, but this proved to be due to the release of nucleosides from an endogenous substrate. 4. The presence of a ;cyclizing' ribonuclease active at alkaline pH on endogenous RNA was confirmed by the inhibitory effects of phosphate, arsenate and bentonite. The activity was unaffected by heat, EDTA or metal ions. The enzyme also acted on exogenous RNA. 5. A purified preparation of neurosecretory granules from fresh bovine posterior pituitary lobes was free from alkaline ribonuclease activity. Most of the activity present in the tissue was recovered in the supernatant plus microsomal material. 6. The distribution of RNA did not follow that of the alkaline ribonuclease.

Animals↗
PubMed1975

The effects of oestradiol-17beta on the ribonucleic acid polymerases of immature rabbit uterus.

Measurements of the endogenous RNA polymerase activities of nuclei isolated from immature rabbit uteri have shown that prior treatment of the animals with oestradiol-17beta has a profound effect on the apparent activities of both RNA polymerases A and B. Within 1 h of hormone treatment, the activity of RNA polymerase A is increased and continues to rise until about 4h when it reaches a plateau and remains steady until at least 8h. The activity of RNA polymerase B increases sharply after oestradiol treatment reaching an early maximum at 30-45 min. Thereafter this activity declines until by 1-2h it approaches control values but a second increase in activity then occurs with a maximum at 3-4h. Treatment of the rabbits with alpha-amanitin before the administration of oestradiol inhibits the hormone-induced stimulation of RNA polymerase A activity in isolated nuclei but when the administration of alpha-amanitin is delayed until after the early rise of RNA polymerase B activity, the oestradiol-induced stimulation of RNA polymerase A is retained. Similar results have been obtained in experiments with cycloheximide suggesting that the stimulation of RNA polymerase A activity by oestradiol is dependent on the hormone-induced stimulation of RNA polymerase B and the subsequent synthesis of protein using the RNA product of the early increase in RNA polymerase B activity. Measurement of the activities of RNA polymerases A and B after isolation of the enzymes from immature rabbit uterine nuclei before and after oestradiol treatment failed to show any differences. Therefore it would appear that the changes in the observed activities of RNA polymerases A and B in isolated nuclei are consequences of changes in the structure and function of chromatin rather than the results of modifications in the RNA polymerases themselves.

Amanitins↗
PubMed1976

Chromatin-bound and free forms of poly(adenylic acid) polymerase in rat hepatic nuclei.

Isolated rat hepatic nuclei were shown to contain poly(A) polymerase in two distinct physiologically active forms. One form was associated with the chromatin fraction and was dependent on endogenous RNA, presumably mRNA. The other activity was localized in the nuclear sap in a 'free' form and was stimulated almost 30-40-fold by exogenously added poly(A). Isolated nucleoli were devoid of significant poly(A)-synthesizing activity.

Animals↗
PubMed1976

Nuclear binding of progesterone in chick oviduct. Multiple binding sites in vivo and transcriptional response.

1. Varied doses of labelled or unlabelled progesterone were injected into immature chicks which had previously been stimulated with oestrogen. The concentrations of nuclear bound [3H]progesterone were correlated with the effects of the hormone on endogenous RNA polymerase I and II activities in isolated oviduct nuclei. 2. The extent of nuclear localization of [3H]progesterone in oviduct (a progesterone target tissue) was shown to be much greater than in lung (non-target tissue). The conccentration of bivalent cations in solvents used in the nuclei isolations has a marked effect on the amount of bound hormone in the nuclei. 3. Evidence for the existence of several classes of binding sites for progesterone in the oviduct nuclei is given. These classes represent about 1000) 10000 and 100000 molecules of the hormone per cell nucleus and are saturated by injecting approx. 10, 100 and 1000 mug of progesterone respectively. 4. When saturation of the first (highest affinity) class of nuclear sites occurs, a marked inhibition in RNA polymerase II (but not RNA polymerase I) activity was observed. When the second class of sites was saturated, alterations in both RNA polymerase I and II activities were observed. Binding to the third class of nuclear binding sites was not accompained by further changes in polymerase activity. It is suggested that the first two classes of nuclear binding sites may represent functional sites for progesterone action in the chick oviduct.

Animals↗
PubMed1978

The use of rat liver nucleoplasm for the characterization of heterogeneous nuclear ribonucleic acid synthesis in vitro.

1. A nucleoplasmic fraction rich in endogenous RNA polymerase II activity was isolated from rat liver nuclei and conditions were determined under which elongation of RNA molecules initiated in vivo continued at maximal rates in vitro. 2. Elongation rates in vitro were calculated to be about 0.25 nucleotide/s and there were about 7 X 10(3) RNA molecules in the process of being elongated by form-II RNA polymerase per original nucleus. 3. Evidence was obtained suggesting that transcription-dependent release of RNA polymerase II molecules from the template occurred during the incubations in vitro. 4. The nascent RNA was tightly associated with protein and banded as ribonucleoprotein in caesium salt gradients. 5. RNA molecules labelled in vitro were up to 13000 nucleotides in length, but consisted of long unlabelled chains transcribed in vivo with only short labelled sequences added in vitro, and without significant polyadenylation. 6. Hybridization of transcripts in the presence of a vast excess of DNA demonstrated that both form-II RNA polymerase and another enzyme, resistant to low alpha-amanitin concentrations, were synthesizing RNA molecules complementary to both reiterated and unique DNA sequences in the genome.

Animals↗
PubMed2002

Mechanism of action of hammerhead ribozymes and their applications in vivo: rapid identification of functional genes in the post-genome era by novel hybrid ribozyme libraries.

A hammerhead ribozyme was demonstrated to be a metalloenzyme. By controlling the metal-binding ability of the hammerhead ribozyme in the presence or absence of a specific sequence of interest, we engineered an allosterically controllable ribozyme, designated the maxizyme. Hybrid ribozymes were then constructed by coupling the site-specific cleavage activity of a hammerhead ribozyme with the unwinding activity of an endogenous RNA helicase. This leads to extremely efficient cleavage of target mRNA, not only in vitro, but also in vivo, and eliminates one of the major problems arising in the application of ribozymes for cleavage of mRNA in vivo : that many target sites on the RNA were previously inaccessible to cleavage owing to secondary and/or tertiary structure formation. Since hybrid ribozymes can efficiently attack target sites within mRNA, libraries were made of hybrid ribozymes with randomized binding arms, which were then introduced into cells. This procedure made it possible to readily identify the relevant genes associated with a specific phenotype, such as in apoptosis and cancer metastasis pathways. This application of a randomized library of hybrid ribozymes represents a simple, yet powerful, method for the identification of genes associated with specific phenotypes in the post-genome era. Moreover, vector-based siRNA (short-interfering RNA for RNA interference, RNAi) can also be used for the creation of the libraries and for the subsequent confirmation of the identified genes, relevant in the examined phenotype.

Gene Expression Profiling↗
PubMed2002

Dynamic response of catabolic pathways to autoacidification in Lactococcus lactis: transcript profiling and stability in relation to metabolic and energetic constraints.

The dynamic response of the central metabolic pathways to autoacidification (accumulation of organic acid fermentation products) in Lactococcus lactis was investigated in a global manner by integrating molecular data (cellular transcript concentrations, mRNA turnover) within physiological investigations of metabolic and energetic parameters. The decrease in pH associated with the accumulation of organic acids modified the physiological state of the cell considerably. Cytoplasmic acidification led to inhibition of enzyme activities and, consequently, to a diminished catabolic flux through glycolysis and a decreased rate of biochemical energy synthesis. This decrease in energy production together with the increased energy expenditure to counter cytoplasmic acidification led to energetic limitations for biomass synthesis. In these conditions, the specific growth rate decreased progressively, and growth ultimately stopped, although a diminished catabolic flux was maintained in the absence of growth. The cellular response to this phenomenon was to maintain significant levels of mRNA of catabolic genes, involving both continued transcription of the genes and also, in certain cases, an increase in transcript stability. Thus, translation was maintained, and intracellular concentration of certain enzymes increased, partially compensating for the inhibition of activity provoked by the diminished pH. When catabolic activity ceased after prolonged exposure to stress-induced stationary phase, endogenous RNA catabolism was observed.

Acids↗