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At least 343 records · Page 19Linked to original sources
PubMed1995

Expression of members of the bcl-2 gene family in the immature rat ovary: equine chorionic gonadotropin-mediated inhibition of granulosa cell apoptosis is associated with decreased bax and constitutive bcl-2 and bcl-xlong messenger ribonucleic acid levels.

The loss of ovarian follicles through atresia, which accounts for greater than 99% of postnatal female germ cell depletion, is mediated through apoptotic cell death. Although recent data have shown that apoptosis in granulosa cells of ovarian follicles can be hormonally manipulated, the molecular mechanisms responsible for transducing external signals remain to be elucidated. Herein we have characterized changes in the expression of the bcl-2 protooncogene (an inhibitor of apoptosis), the bax gene (an inducer of apoptosis), and the bcl-x gene (which encodes both bcl-xlong, an inhibitor of apoptosis, and bcl-xshort, an inducer of apoptosis) during gonadotropin-stimulated follicular development in vivo and during atresia of antral follicles incubated in vitro. Complementary DNA fragments corresponding to rat bcl-2, rat bax, and rat bcl-x coding sequences were obtained by the reverse transcription-polymerase chain reaction (RT-PCR) technique using total RNA prepared from immature rat ovaries. Northern blot analysis of steady-state bcl-2, bax, and bcl-x messenger RNA (mRNA) levels in total RNA prepared from ovaries of immature rats before and after in vivo priming with 10 IU equine CG (eCG) revealed that eCG-induced follicular growth and survival were associated with a relatively constitutive level of bcl-2 and bcl-x expression but markedly reduced levels of bax mRNA (29 +/- 5% of saline-treated control animals). Using the RT-PCR technique coupled with Southern blot hybridization analysis to distinguish the long vs. short forms of bcl-x (which differ in size by 189 base pairs), it was determined that bcl-xlong was the predominant message expressed by granulosa cells of eCG-primed ovaries. The eCG-mediated decrease in bax expression, coupled with a maintenance of bcl-2 and bcl-xlong mRNA levels, were associated with a pronounced reduction in the extent of granulosa cell apoptosis. In contrast, the induction of apoptosis in a homogeneous population of healthy antral follicles by incubation in vitro without tropic support was associated with an significant increase in bax mRNA levels to 220 +/- 10% of those detected in freshly isolated, healthy follicles. Moreover, bcl-xlong message levels were significantly reduced in follicles incubated for 24 h to 69 +/- 4% of those levels detected in freshly isolated, healthy follicles. However, no significant change in the level of bcl-2 mRNA was detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1997

Peroxisome proliferator-activated receptor gamma1 expression is induced during cyclic adenosine monophosphate-stimulated differentiation of alveolar type II pneumonocytes.

The primary function of lung alveolar type II cells is to synthesize pulmonary surfactant, a lipoprotein enriched in dipalmitoylphosphatidylcholine. Because type II pneumonocytes are highly lipogenic, we considered the possible role of the adipogenic nuclear hormone receptor, peroxisome proliferator-activated receptor gamma (PPARgamma), in their differentiation from epithelial cell precursors. A degenerate PCR-screening strategy revealed that multiple PPARs, including PPARgamma, are present in differentiated type II cells. A PCR-amplified PPARgamma DNA-binding domain was used to isolate a full-length PPARgamma1 complementary DNA clone from a rabbit type II cell complementary DNA library. Although another PPARgamma isoform, PPARgamma2, is known to be highly expressed in adipocytes, only PPARgamma1 was detected in rabbit type II cells by use of RT-PCR and by library screening. Rabbit PPARgamma1 has 90% nucleotide sequence identity and 95% amino acid identity to mouse PPARgamma1. PPARgamma1 messenger RNA was readily detected in total RNA isolated from rabbit type II pneumonocytes cultured in the presence of cAMP, which causes enlargement of the prealveolar ducts, accelerates the rate of type II cell differentiation, and induces transcription of the major surfactant associated protein, surfactant protein-A. PPARgamma1 messenger RNA also was detected in total RNA isolated from rabbit adipose tissue but not from whole adult or fetal lung, heart, or liver. By Western blot analysis, PPARgamma protein expression was found to occur coincidentally with surfactant protein-A expression during lung type II cell differentiation. In view of the role of PPARgamma in adipocyte differentiation and lipid homeostasis, we postulate that PPARgamma1 induction by cAMP plays a role in the differentiation and expression of lipogenic enzymes in lung type II cells.

Adipose Tissue↗
PubMed1975

Decay of ribosomal ribonucleic acid in Escherichia coli cells starved for various nutrients.

Decay of pre-existing ribonucleic acid was studied in Escherichia coli cells subjected to high temperature or to starvation for nitrogen, phosphate, amino acids, or a carbon source. In these studies a series of mutants affected in ribonucleic I(RNase I, EC 3.1.4.22) polynucleotide phosphorylase (EC 2.7.7.8) or ribonuclease II (RNase II, EC 3.1.4.23) were used. Degradation of total RNA and the disappearance of 23 S and 16 S rRNA were followed. The results obtained indicated that, by and large, decay of 23 S and 16 S RNA parallels that of total RNA. Decay of RNA depended on the nuclease content of the cells as well as on the treatment of applied. It was most pronounced during carbon starvation and least in cells deprived of phosphate ions. It was most effective in strains containing all three nucleases and least in the strain defective in all three. The exonucleases polynucleotide phosphorylase and RNase II did not seem to affect the extent of 23 S and 16 S RNA disappearance. Strains with modified exonucleases did accumulate low molecular weight RNA species during treatments which induced considerable degradation of 23 S and 16 S RNA. Based on the above date and previous observations, we suggest that during various starvations a similar mechanism is operative. The 23 S and 16 S RNAs are degraded endonucleolytically, and this is the rate-limiting step during starvation. The exonucleases polynucleotide phosphorylase and RNase II seem to participate primarily in the decay of the low molecular weight RNA species formed by the endonuclease(s), not as yet identified.

Amino Acids↗
PubMed2004

Tick-borne encephalitis virus-specific RT-PCR--a rapid test for detection of the pathogen without viral RNA purification.

Among diseases transmitted by ticks in the Czech Republic, tick-borne encephalitis (TBE) caused by Tick-borne encephalitis virus (TBEV) and Lyme disease caused by Borrelia burgdorferi spirochete are most important. We propose an effective and specific test for detection of TBEV in a single tick or a pool of ticks based on the detection of TBEV RNA using an RT-PCR technique without RNA purification. The method is very sensitive with the detection limit of about 14 fg TBEV RNA in total RNA obtained from brain suspension from suckling mice infected with TBEV per reaction. The primers were derived from the 5'-terminal non-coding region, a highly conserved part of the virus. The method was successfully applied to field-collected ticks in detecting TBEV RNA. This method can be used in studies of several aspects of TBEV: epidemiology, screening of natural foci, circulation and detection of virus genome sequences in clinical materials.

5' Untranslated Regions↗
PubMed1988

Cell-free synthesis of a putative precursor to the rat liver mitochondrial glycerol-3-phosphate dehydrogenase.

Antibodies to purified glycerol-3-phosphate dehydrogenase were raised in rabbits and purified from serum by affinity chromatography on enzyme-bound Sepharose columns. RNA from membrane-free polyribosomes, or poly(A)+ RNA (total cellular RNA) of rat liver, was translated in a rabbit reticulocyte protein-synthesizing system in the presence of [35S]methionine, and the glycerol-3-phosphate dehydrogenase synthesized was isolated by immunoprecipitation using the antibody. The in vitro product moved on sodium dodecyl sulfate-polyacrylamide gels as a polypeptide that was about 5,000 daltons larger than the subunit of the mature enzyme (74,000 daltons). Digestion of both the mature and the in vitro newly synthesized forms of the enzyme yielded respective sets of peptide fragments which had similar patterns upon sodium dodecyl sulfate-gel electrophoresis. When the presumptive precursor that had been synthesized in vitro was incubated with isolated intact rat liver mitochondria, it was converted to "mature" subunits that were no longer susceptible to externally added proteases. Import of the presumptive precursor is dependent upon an electrochemical potential across the inner mitochondrial membranes. The mature form of the protein is assembled in its native location (the outer surface of the inner mitochondrial membrane).

Animals↗
PubMed1978

[Biospecific chromatography of poly(A)-containing RNA on poly(U)-Sepharose].

It is shown that in addition to specific binding of polyadenylic sequence with poly(U), the chromatography of poly(A)-containing RNAs on poly(U)-Sepharose is accompanied by nonspecific irreversible adsorption of polynucleotides on Sepharose gel. This disadvantage may be overcome by establishing optimal BrCN/Sepharose rations during Sepharose activation and by many-fold treatment of poly(U)-Sepharose with ethanolamine immediately before chromatography of RNAs. It was also found that the efficient separation of poly(A+)-RNA preparations from poly(A-)-RNAs is achieved only after double chromatography of RNA on poly(U)-Sepharose. The amount of poly(A+)-RNA in total RNA preparations isolated from bound polyribosomes of 10-day-old chick embryos is equal to 1%. Data from PAAG gel electrophoresis are indicative of the lack of degradation and high heterogeneity of the preparations under study.

Chemical Phenomena↗
PubMed1993

Are chimeric kainate/N-methyl-D-aspartate receptors expressed in Xenopus oocytes from mammalian and amphibian RNA?

The Xenopus oocyte expression system has been used to test for the existence in rat and Xenopus central nervous systems (CNS) of excitatory amino acid receptors (EAAR) that show sensitivity to both N-methyl-D-aspartate (NMDA) and L-kainate. Two to 15 days after injection of oocytes with rat brain poly(A+) RNA (or total RNA) and Xenopus CNS poly(A+) RNA, voltage-clamped oocytes were tested for EAAR expression by exposure to various excitatory amino acids (EAA). Responses to coapplication of NMDA and/or kainate were less than the sum of those obtained during application of each of these two EAA alone. In both cases, the antagonism was noncompetitive, although at millimolar concentrations, competitive antagonism was also observed. The extent of noncompetitive antagonism by NMDA of the response to kainate was proportional to an oocyte's sensitivity to NMDA. Oocytes injected with mRNA transcribed from the GluR1 clone encoding a rat non-NMDA receptor subunit did not respond to NMDA, and antagonism of the response to kainate occurred only with millimolar concentrations of NMDA and was competitive. Various explanations for these results are discussed. One possibility is that Xenopus oocytes injected with these vertebrate CNS RNA express a subpopulation of receptors containing kainate-sensitive and NMDA-sensitive subunits in addition to NMDA receptors and non-NMDA receptors. This explanation is supported by the observation that the NMDA antagonists MK-801 and magnesium antagonize part of the current induced by kainate, but only when NMDA is present.

Animals↗
PubMed1994

Nucleotide sequence of the proton ATPase beta-subunit homologue of the sea urchin Hemicentrotus pulcherrimus.

A cDNA with 2.3 kb encoding F1-F0 ATP synthase (proton ATPase) beta-subunit homologue was isolated from a testis cDNA library of the sea urchin, Hemicentrotus pulcherrimus. The deduced amino acid sequence consisted of 523 residues which contained a 19-residue amino-terminal signal peptide and a 8-residue glycine-rich consensus sequences. Analysis of poly(A) +RNA and/or total RNA from H. pulcherrimus testis, ovary, unfertilized eggs, and embryos by Northern blot revealed a 2.4 kb RNA.

Amino Acid Sequence↗
PubMed1998

mRNA levels of alpha1(VI) collagen, alpha1(XII) collagen, and beta ig in rabbit cornea during normal development and healing.

PURPOSE: Type VI and XII collagens and beta ig, major components of the interfibrillar matrix, may maintain proper spacing among collagen fibrils, necessary for corneal transparency. During normal corneal stroma development and healing, changes in mRNA levels of these proteins were measured to determine whether differences in steady state levels are indicative of the unique structure produced by each corneal tissue. METHODS: A full-thickness excision wound was made in each cornea of six adult rabbits and allowed to heal for 1, 2, or 4 weeks. Scar tissue from two rabbits (four scars) were used from each time period and processed for RNA extraction. Total RNA from 23-day-old fetal rabbit corneas (equivalent to approximately 1 week of stromal development) and 8-day-old neonate corneas (equivalent to approximately 3.5 weeks of stromal development) was also extracted. Relative quantities of alpha1(VI) collagen, alpha1(XII) collagen, beta ig, and beta-actin mRNAs were determined by competitive reverse transcriptase-polymerase chain reaction. Glyceraldehyde-3-phosphate dehydrogenase was used as a housekeeping gene. RESULTS: Increased mRNA levels for alpha1(VI) and alpha1(XII) collagens, beta ig, and beta-actin were seen during the first 2 weeks of healing and were followed by a decrease in 4-week-old scars. Similar increases were seen in fetal corneas with a further increase in the neonate. Differences in the beta ig mRNA levels relative to that of alpha1(XII) collagen in fetal stroma and in comparison with 1-week-old wounds suggest a higher production of beta ig in early healing tissue. CONCLUSIONS: Alterations of mRNA levels during healing and development are consistent with the cellular events and deposition of extracellular matrices in these corneal tissues. Assuming that extracellular matrix protein production is regulated at the transcriptional level, relative changes in beta ig and collagen mRNA levels reflect differences in protein deposition in early fetal and healing tissues. This is consistent with differences in the organization of the interfibrillar matrices of these tissues and their transparency.

Actins↗
PubMed2005

Validation and application of a high fidelity mRNA linear amplification procedure for profiling gene expression.

The need for microgram quantities of RNA for microarray experiments has hindered application of this novel technology in cell types/tissue samples with limited abundance of RNA. In this study, potential application of T7-based linear RNA amplification was investigated for use in gene expression profiling experiments where starting material is limited. Yield and integrity of amplified antisense RNA (aaRNA), microarray hybridization intensities, and fidelity of differential gene expression detected were determined for arrays generated for unamplified versus amplified RNA from the same homogenous starting pools. Total RNA was extracted from bovine spleen and fetal ovary, serially diluted to concentrations ranging from 2 microg to 500 pg and amplified. Quality and quantity of total input RNA and aaRNA were assessed by spectrophotometry, gel electrophoresis and bioanalyzer. In experiment 1, we determined the optimal amounts of aaRNA generated from 20, 40, 200 ng and 2 microg input total RNA for use in cDNA synthesis, labeling and array hybridization that would yield robust and consistent hybridization signals on a bovine oocyte cDNA microarray. In experiment 2, comparison of microarray hybridization intensities and fidelity of differential gene expression between aaRNA generated from 2, 20 and 40 ng input total RNA versus unamplified RNA (uRNA) were conducted. The hybridization intensities for each of the 7000 spots per slide for microarrays conducted using aaRNA versus uRNA were highly correlated (2 ng = 0.84, 20 ng = 0.88, 40 ng = 0.90; P < 0.01). The false positive rate was low and similar (4.0% versus 4.4%) for arrays done with uRNA and aaRNA. Ninety-seven ESTs were detected as differentially expressed in the fetal ovary versus spleen at > 1.5- or < 0.5-fold using uRNA (P < 0.05). However, the number of genes detected in arrays using aaRNA was approximately 1.5-2.5 times greater than with uRNA. Approximately, 65-70% of differentially expressed genes were common between uRNA and aaRNA arrays. Relative fold-expression (Cy3/Cy5 ratios) for 25 overlapping abundant genes was comparable for uRNA versus aaRNA arrays with 2 and 20 ng total RNA as input. Results demonstrate that T7-based linear amplification of small amounts of input RNA and use of aaRNA in microarray experiments retains fidelity of detection of differential gene expression that is relatively comparable to experiments done with uRNA and provides a potentially viable approach to facilitate gene expression profiling using limited amounts of starting material.

Animals↗
PubMed1996

Co-ordinate induction of collagen type I and biglycan expression in keloids.

Proteoglycans are macromolecules displaying structural roles as well as regulatory functions in the maintenance of the extracellular matrix. Biglycan/PG-I and decorin/PG-II are two small proteoglycans that are structurally related but differ considerably in their localization in vivo and behaviour in vitro. Decorin and, to a minor extent, biglycan, can be located at the surface of type I collagen fibrils and have been shown to influence collagen fibrillogenesis. However, the physiological role of biglycan in the dermis is not known. Biopsies obtained from keloids were bisected and processed for total RNA extraction and immunohistochemistry. Northern blot analysis of total RNA obtained from keloids with high growth tendency in vivo showed a marked induction of biglycan and collagen alpha 1(I)mRNA expression in comparison with total RNA obtained from normal skin or keloids with little growth tendency. In contrast, decorin mRNA expression remained largely unaltered. Studying these biopsies by immunohistochemistry, decorin expression in the dermis was unaltered comparing normal and keloid tissue, whereas a markedly increased staining for biglycan was observed in the keloid tissue, which was most pronounced in the nodular formations, and was a characteristic feature of keloids. The altered expression of biglycan in keloid tissue might be involved in the abnormal regulation of extracellular matrix deposition either through the binding of growth factors or by influencing the three-dimensional organization of collagen fibres or associated molecules.

Biglycan↗
PubMed2002

Intragraft messenger RNA expression of angiotensinogen: relationship with transforming growth factor beta-1 and chronic allograft nephropathy in kidney transplant patients.

Transforming growth factor (TGF)-beta1 is important in fibrogenesis and has been involved in the pathogenesis of chronic allograft nephropathy (CAN). The angiotensinogen (AGT) gene encodes the only glycoprotein known to be a precursor of the vasopressor angiotensin II. Angiotensin II is also a growth factor and a profibrogenic cytokine. It mediates the induction of TGF-beta1. We studied the relationship among the intragraft expression of AGT, TGF-beta1, and CAN in stable renal transplant patients (RTP). We used a competitive quantitative reverse transcriptase-polymerase chain reaction (RT-PCR)-ELISA assay to identify intragraft amounts of AGT expression in RTP and correlated it with TGF-beta1 mRNA expression. We studied and performed kidney biopsies on 12 RTP with long-functioning grafts and 6 RTP in the immediate posttransplantation period (7 days) who had acute tubular necrosis as control. Histology was based on Banff working classification criteria. Total RNA was isolated from biopsy specimens. For RT-PCR-ELISA, we created heterologous RNA competitors that coamplified with the same primers as AGT and TGF-beta1. Six of 12 long RTP had proteinuria >1000 mg/24 hr and 6 had proteinuria <1000 mg/24 hr. The differences between Banff grades (P =0.03), AGT, and TGF-beta1 levels by RT-PCR-ELISA were statistically significant between both groups (106.2+/-60.7 vs. 34.1+/-11.9 pg/microg total RNA [P =0.01] and 5954+/-5612 vs. 436+/-517 transcripts/microg total RNA [P =0.01], respectively). The control group showed AGT levels of 25+/-12.2 pg/microg total RNA and TGF-beta1 levels of 228+/-111 transcripts/microg total RNA, significant only for the higher proteinuria group (P=0.01 and P=0.04, respectively). There was a correlation between AGT and TGF-beta1 in both groups (r=0.96, P=0.001). We showed a relationship between mRNA expression of AGT and TGF-beta1 in kidney transplant patients with different grades of CAN and proteinuria.

Adult↗
PubMed1990

Ontogeny and pituitary regulation of testicular growth hormone-releasing hormone-like messenger ribonucleic acid.

The testis is rich in central nervous system-type neuropeptides, including a GH-releasing hormone (GHRH)-like substance. We examined the ontogeny and pituitary regulation of testicular GHRH-like mRNA (t-GHRH mRNA) and compared this to expression of insulin-like growth factor-I (IGF-I) and IGF-II mRNA in developing testis. t-GHRH mRNA was measured by dot blot hybridization and quantitated using a hypothalamic GHRH cRNA standard. t-GHRH mRNA was not detectable in Northern blots in fetal testis on day 19 of gestation, but was present in low but detectable amounts in testicular dot blots on day 2 of life (0.44 pg/micrograms total RNA). Levels of the RNA increased beginning on day 21 (1.72 +/- 0.23 pg/micrograms total RNA) and reached adult levels by day 30 (4.96 +/- 0.84 pg/micrograms total RNA). The GHRH species on Northern analysis was about 1750 nucleotides at all ages examined; there was a larger species of about 3350 nucleotides seen on days 65 and 90. There was no correlation between the ontogeny of t-GHRH mRNA and either IGF-I or IGF-II mRNAs, which were maximally expressed in the testes of day 2 animals and decreased with age. To examine the influence of the pituitary gland on t-GHRH mRNA, levels of the mRNA were measured in the tests of hypophysectomized animals and age-matched controls. In animals hypophysectomized on day 21 and killed on day 42 and in animals hypophysectomized on day 42 and killed on day 63, there was marked diminution of t-GHRH mRNA (19 +/- 5% and 9 +/- 2% of age-matched controls, respectively). In contrast, in animals hypophysectomized on day 65 and killed on either day 80 or 90, there was a much smaller difference in levels of t-GHRH mRNA compared to values in control animals (73 +/- 20%). This was unlike the effect of hypophysectomy on testicular IGF-I mRNA, where uniform diminution was seen in all three groups. Because GH is important in the regulation of hypothalamic GHRH mRNA, we examined the effects of administration of recombinant human GH on the reinduction of t-GHRH mRNA after hypophysectomy and compared this to the reinduction of IGF-I mRNA. Neither t-GHRH mRNA nor testicular IGF-I mRNA increased in hypophysectomized animals treated with GH. Our results indicate that t-GHRH mRNA is developmentally regulated, and that the hypothalamic-pituitary axis is important in its expression.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2005

[Cloning of Human canstatin gene and expression of its recombinant protein].

OBJECTIVE: To clone human canstatin gene and express its recombinant protein. METHODS: The total RNA was extracted from human placenta. The canstatin gene fragment was synthesized and amplified from the total RNA by RT-PCR. The resulting product was cloned into pUCm-T vector and transformed into E.coli DH5alpha through electroporation. The gene was sequenced by the Sanger Dideoxy-mediated chain-termination method, and then the canstatin cDNA was cloned into the BamHI and HindIII sites of plasmid pET-22b (+) and transformed into E.coli BL21 where it was induced to express proteins by isopropyl-1-thio-b-Dgalactopyranoside (IPTG). RESULTS: The extracted total RNA was separated into three clear bands indicating 28S, 18S, and 5S after electrophoresis. The canstatin gene fragment was synthesized and amplified from the total RNA by RT-PCR. The resulting products were cloned into pUCm-T vectors, and then were transformed into E.coli DHSa. After an over night culture, both blue and white colonies were found on the agar plate. Six white colonies were selected and cut by BamHI and HindIII. The plasmids DNA in one white colony showed one band near the location of primary plasmid after digested by BamHI and two bands near the locations of primary plasmid and objective gene fragment after digested by HindIII. The cloned gene in this white colony was sequenced and demonstrated to have the same sequence as that of canstatin gene in GenBank. Then canstatin cDNA was cut down from pUCm-T with BamHI and HindIII and ligated into the vector pET-22b (+). The resultant plasmid pET-22b (+)/canstatin was then transformed into E.coli BL21. White colonies were found on LB agar plate. Seven of them were selected and their plasmids were digested with both BamHI and HindIII. After electrophoresis, all selected colonies showed two specific bands, one was found near the location of primary plasmids, and the other near that of objective gene fragment. After IPTG induction, there was a new protein band about Mr 24 000 on SDS-PAGE. As estimated by densitometry, the percentage of the expressed product over total bacterial proteins was 18.2%, 18.8%, 23.0% and 23.4%, respectively, 1, 2, 3, and 4 hours after induction. CONCLUSION: Human canstatin gene was successfully cloned and its recombinant proteins were expressed in this study.

Base Sequence↗
PubMed2000

Androgen regulation of the largest subunit of RNA polymerase II in the rat ventral prostate.

One of the dramatic changes in the prostate during androgen manipulation is the alteration in cellular content of total RNA - the amount of total RNA in each cell. The abundance of cellular total RNA correlates with the RNA polymerase (RNAP) activity in the prostate. One possible mechanism of androgen regulation of RNAP activity involves the regulation of RNAP expression. Western blot analysis showed that the largest subunit of the RNAP II, an essential component of the transcriptional machinery for mRNA, is indeed regulated by androgens. Castration down-regulates the protein level of RNAP II, whereas androgen replacement up-regulates the protein. However, androgen manipulation does not have consistent effects on the phosphorylation of the C-terminal domain (CTD) of the RNAP II. Androgen regulation of the RNAP II protein expression was also observed in the seminal vesicles but not in the thymus and liver, indicating that androgen regulation of RNAP II protein expression appears to be limited to the male sex accessory organs. These observations suggest that RNAP II plays an essential role in androgen action in male sex accessory organs.

Androgens↗
PubMed1974

Translation in vitro of total nuclear RNA from HeLa cell nuclei infected with adenovirus 2.

Total heterogeneous nuclear RNA from HeLa cells infected with adenovirus for 18-20 hr stimulates amino acid incorporation into protein in a cell-free system from Ehrlich ascites tumors. This stimulation of protein synthesis by the nuclear RNA requires intact nuclei. The role of nuclei in this system is unknown, but evidence is presented that the nuclei are involved in the conversion of high-molecular-weight RNA to low-molecular-weight species. Some of the newly synthesized polypeptides appear to resemble the virion polypeptides.

Adenoviridae↗
PubMed1997

RNase Activities Are Reduced Concomitantly with Conservation of Total Cellular RNA and Ribosomes in O2-Deprived Seedling Roots of Maize.

The effect of O2 deprivation on the activities of RNases and levels of total cellular RNA and ribosomes in seedling roots of maize (Zea mays L.) was investigated. Sodium dodecyl sulfate-polyacrylamide gels containing RNA were used to distinguish RNase isoenzymes by apparent molecular mass. Since O2 deprivation causes a decrease in cytosolic pH from approximately pH 7.4 to 6.4 and an elevation in cytosolic Ca2+, RNase levels were examined in the physiological range of cytosolic pH and in the presence of Ca2+, Mg2+, Zn2+, ethylenediaminetetracetate, or ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime]-tetraacetic acid. The activity of a number of RNases present in aerobic roots was reduced in response to O2 deprivation. Several RNases with a pH optimum of 6.4 were rapidly down-regulated by O2 deprivation. Spectrophotometric assay of extracts revealed that RNase activity was higher at pH 6.4 than at 7.2, and ethylenediaminetetracetate-insensitive RNase activity decreased in response to O2 deprivation. The decrease in RNase activity was correlated with no loss of total cellular RNA or ribosomes, despite a 4-fold decrease in run-on transcription of rRNA in isolated nuclei. Regulation of RNase activity may facilitate the conservation of nontranslating ribosomes and poorly translated mRNAs during O2 deprivation.

Journal Article↗
PubMed1978

RNA synthesis in whole cells and protoplasts of centaurea: a comparison.

Protoplasts enzymically isolated from suspension cultures of Centaurea cyanus L. incorporate radioactive precursors into RNA with kinetics similar to that of whole cells. There are differences, however, in several other aspects of RNA metabolism. The proportion of total RNA that contains poly(A) sequences (25 to 30%) is similar in both freshly isolated protoplasts and whole cells after a 20-minute pulse with [(3)H]adenosine. After a 4-hour pulse, however, poly(A)-containing RNA makes up 30% of the total RNA in protoplasts whereas it drops to 8% in whole cells. There appears to be a faulty processing of ribosomal precursor into the mature ribosomal species, as the precursor seems to accumulate to higher levels relative to the mature 18S and 25S rRNAs in protoplasts as compared to whole cells. Additional differences are seen in the size distributions of poly(A)-containing RNA, although the length of the poly(A) segment is similar in both protoplasts and whole cells. Within 24 hours protoplasts appear to have resumed a pattern of RNA synthesis similar to that of whole cells.

Journal Article↗