Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.5 (RNase)
 17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Five ribonuclease activities, separable by polyacrylamide gel electrophoresis, have been detected in erythroid bone marrow cells from anaemic rabbits. Their intracellular distribution has been investigated and compared with that of the ribonucleases in reticulocytes. Both the acid and alkaline ribonuclease activities of reticulocytes are much lower (30--50 fold) than those of bone marrow erythroid cells. The most marked decrease in enzyme activity occurs in the fractions containing ribosomes and mitochondria plus lysosomes. In these subcellular organelles there was also a qualitative change in the ribonuclease electrophoretic pattern, whereas the cytosol enzymes of marrow erythroid cells and reticulocytes remained largely unchanged. Several ribonucleases released from reticulocyte membranes with urea were similar to those present in the lysosomal plus mitochondrial fraction, as shown by detection of enzyme activity after polyacrylamide gel electrophoresis. The decline in ribonuclease activity was found to begin in the orthochromatic cells, which have a highly condensed nucleus and are no longer active in DNA and RNA synthesis, and to coincide with a decrease in acid phosphatase activity and loss of lysosomes.
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PMID:Intracellular distribution of ribonuclease activity during erythroid cell development. 1 51

Precursor mRNA (pre-mRNA) was extracted from erythroid enriched bone marrow cells of the rabbit by the methods of Georgiev and Mantieva modified by Markov and Arion and of Holmes and Bonner, respectively. Density gradient centrifugation, base analysis and the effects of alpha-amanitin and actinomycin D on the synthesis of the cellular RNA showed signs of degradation in the rRNA-free 85 degrees C-fraction of the preparation according to Georgiev and Mantieva and a substantial rRNA contamination of the 65 degrees C-fraction. This RNA-fraction as well as the total RNA-preparation extracted according to Holmes and Bonner was purified from rRNA by affinity chromatography on poly(U)-Sepharose. Poly(A)+-RNA of all size-classes, among it a substantial amount of high molecular weight RNA (greater than 45 S), was isolated by this purification procedure. Especially the extraction according to Holmes and Bonner yields high molecular weight material but the critical step of this procedure often resulting in degradation of the RNA is the DNase treatment of the heavily DNA-contaminated total RNA-preparation either due to RNase contamination of the DNase or to the existence of RNase in the less intensive deproteinized RNA. The investigated cellular system is characterized by a very intensive rRNA synthesis which is typical for cells in the early stages of hematopoiesis. In contrast to investigations with purified RNA-polymerases and subcellular systems, but in accordance with data of in vivo experiments, alpha-amanitin inhibits both the pre-mRNA and the pre-rRNA synthesis.
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PMID:Pre-mRNA from erythroid enriched bone marrow cells of the rabbit. II. Characterization of pre-mRNA isolated by phenol extraction and poly(U)-Sepharose chromatography. 74 68

Pre-mRNA from bone marrow of rabbits enriched in erythroid cells was analyzed by T1 and pancreatic RNase treatment and poly(U)- and poly(A)-Sepharose chromatography to contain poly(A)-, oligo(U)- and double stranded sequences. The length of the poly(A)- and oligo(U)-sequences was determined by polyacrylamide gel electrophoresis using poly(A)- and oligo(U)-standards of defined length. Poly(A) from poly(A)+pre-mRNA isolated according to the method of Holmes and Bonner shows a size distribution between 40 and 130 nucleotides with an average of 75 nucleotides. Hot phenol extraction according to Georgiev et al. leads to a smaller size of about 25 nucleotides. The oligo(U)-segment consists of 80% U and is about 25 nucleotides long. Poly(A)+ pre-mRNA of about 12000--16000 nucleotides posseses 1--2 oligo(U)-units and one double strand of about 70 nucleotide pairs. Most (greater than 90%) of the oligo(U)-and the double stranded sequences are localized at least 1700 nucleotides away from the 3'terminus. Double strands were investigated with respect to their reannealing behaviour. The material consists of two types of double strands: 20% which reassociate at a cot/2 cot/2 of 1.3 . 10(-4) represent only one or a few types of double strands, the remaining 80% reassociate at a cot/2 of about 7 . 10(-2) and are more complex. Under hybridization conditions pre-mRNA molecules are able to self-annealation. 10% of the sequences become RNase stable.
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PMID:Pre-mRNA from erythroid enriched bone marrow cells of the rabbit. III. Poly(A)-, oligo(U)- and double stranded sequences. 74 69

Poly(A)-containing mRNAs labeled with [methyl-3H]methionine were isolated from nucleated erythroid cells obtained from the spleens of anemic mice. The RNAs were further separated into non-globin poly(A)-containing RNAs and highly purified globin mRNA by globin cDNA-cellulose affinity chromatography. DEAE-Sephadex column chromatography of the T2 ribonuclease digestion products of the cDNA-purified globin mRNA fraction yielded methylated resistant fragments with charges of -4.7 (Cap 1) and -5.3 (Cap 2). Digestion of the non-globin RNA fraction revealed a similar pattern with the addition of a methylated mononucleotide identified as 6-methyladenosine at -2 charges. Alkaline phosphatase treatment of the T2 resistant fragments reduced their charges by approximately 2, which is consistent with the removal of one terminal phosphate. Treatment of the globin T2 and alkaline phosphatase-resistant fragments withpenicillium P1 nuclease and alkaline phosphatase yielded a P1-resistant core structure in both fragments. In addition to the core, 2'-O-methylcytidine (Cm) was released from the more negatively charged globin fragment. The P1-resistant cores of the cap structures eluted from DEAE-Sephadex with the known standard m2G5'ppp5'Am and were found to be pyrophosphatase-sensitive establishing a 5'-5'-triphosphate linkage. The pyrophosphatase and alkaline phosphatase digestion products of the globin Cap 1 and Cap 2 core structures were analyzed by high voltage electrophoresis and paper chromatography and found to be 7-methyiguanosine (m7G) and the dimethylated nucleoside 6-methyl-2'-O-methyladenosine (N6mAm). A small amount of the singularly methylated adenosine, 2'-O-methyladenosine (Am) was also observed. The predominant sequences of the methylated nucleosides in the globin cap structures are therefore m7G5'ppp5'N6mAm and m7G5'ppp5'N6mAmpCm.
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PMID:Methylated nucleosides in globin mRNA from mouse nucleated erythroid cells. 83 41

Poly(A)-containing messenger RNA isolated from rabbit reticulocytes as estimated by periodate oxidation and condensation with [3H]isoniazid has two oxidizable end groups per molecule of mol. wt. 220000. When the mRNA is subjected to stepwise degradation by beta-elimination, only one oxidizable end-group is found. This indicates that one of the 2',3' hydroxyl end-groups is linked through the normal 3'--5' phosphodiester bond, but that the other is linked in such a way that after stepwise degradation no new 2',3 hydroxyl group is revealed. This structure could be a 5'-linked 5'-phospho di- or tri-ester. On digestion with ribonuclease the isoniazid-labelled RNA produced oligonucleotide hydrazones consistent with a poly(A) sequence at the 3' end plus fragments that are not found after stepwise degradation. These fragments have a charge of --6 and --8 from pancreatic ribonuclease or --7 from ribonuclease T1 digestion. These charges are changed to --3.4 and --4.1 after pancreatic ribonuclease, ribonuclease T2 and alkaline phosphatase digestion. methyl-3H-labelled-poly(A)-containing RNA isolated from late erythroid cells contain a methyl-labelled fragment resistant to endonuclease and phosphodiesterase II digestion. After digestion with phosphodiesterase I this fragment produces methyl-3 H-labelled nucleotides with the electrophoretic mobility of pm7G and pAm. It is concluded that globin mRNA has the 5' sequences m7G(5')ppp'AmpYpGp ... and m7G(5')pppAmpApGpYp.
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PMID:The nature of the 5'-linked 5' nucleotide sequence at the 5' end of rabbit globin messenger ribonucleic acid. 94 25

Nucleated erythroid cells isolated from the spleens of anemic mice were used to investigate the processing of the polyadenylic acid region of globin mRNA. Cells were labeled in media containing [3H] adenosine and transferred to media containing no radioactive precursor and incubated further in the presence or absence of actinomycin D. After various times following the transfer of the cells, globin mRNA was isolated using a combination of oligo(dT)-cellulose affinity chromatography, sucrose density centrifugation, and globin cDNA (the complementary DNA copy of globin mRNA)-cellulose affinity chromatography. The size of the poly(A) region was determined by polyacrylamide gel electrophoresis of the T1 and pancreatic RNase-resistant fragments. The prelabeled poly(A) region which initially comprises approximately 150 adenylate residues was found to become shorter with time, both in cells incubated in medium containing no radioactive precursor and in the presence of actinomycin D. After 9 h of incubation in the presence of actinomycin D, two major size classes of poly(A) were observed, one containing 35 to 45 adenylic acid residues and the other containing 55 to 65 residues. These two size classes are similar to those found in circulating reticulocytes suggesting that the poly(A) shortening observed in these cell incubation studies is similar to that which occurs in vivo. Two protein synthesis inhibitors, emetine and cycloheximide, were investigated with respect to their effect on poly(A) shortening. Neither drug inhibited the shortening of the poly(A) region of globin mRNA, suggesting that protein synthesis is not required for this process to occur.
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PMID:Shortening of the poly(A) region of mouse globin messenger RNA. 96 74

The size of the polyadenylate segment of globin messenger RNA isolated from spleens of anaemic rabbits was estimated by comparison of its electrophoretic migration in polyacrylamide gels to that of synthetic poly(A) segments of known lengths. Conditions of enzymic degradation of mRNA with pancreatic ribonuclease and T1 ribonuclease were carefully established in order to ensure complete degradation of the heteropolymeric part of mRNA without affecting the polyadenylate sequence. The poly (A) segments of spleen globin mRNA were found to be 25-90 nucleotides long whilst those of peripheral blood reticulocytes from the same animals were only 10-30 residues long. Since spleen contains young erythroid cells and since anucleated blood reticulocytes constitute a statistically older population of the same cell line, these results support the idea that the poly(A) segment of mRNA shortens when the message ages.
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PMID:Globin messenger RNA from anaemic rabbit spleen. Size of its polyadenylate segment. 97 66

The size of the polyadenylic acid region of newly synthesized globin mRNA was determined on mRNA isolated from nucleated erythroid spleen cells of anemic mice. The globin mRNA was purified by a combination of affinity chromatography on oligodeoxythymidylate-cellulose (oligo-(dT)-cellulose) and sucrose density gradient centrifugation. The purified RNA was shown to be globin mRNA by virtue of its ability to direct the synthesis of mouse alpha- and beta-globin chains in a cell-free system and by the presence of two bands migrating identically with authentic mouse alpha- and beta-globin mRNA when subjected to electrophoresis in polyacrylamide gels in the presence of formamide. When labeled for 1 hour with [3H]adenosine, the newly synthesized radioactive mRNA also migrated as two bands in these gels but they moved slower than the main bands suggesting that they have higher molecular weights. The polyadenylic acid region of the mRNA was isolated from the T1 and pancreatic RNase digestion mixture by acrylamide sodium dodecyl sulfate gel electrophoresis. The polyadenylic acid region was found to contain approximately 150 adenylate residues. As it is known that globin mRNA isolated from reticulocytes contains only 40 to 60 residues, it follows that at least 150 adenylic acid residues are added to the globin mRNA soon after its synthesis and that some of these are removed during the subsequent maturation of the erythroid cell.
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PMID:Size of the polyadenylic acid region of newly synthesized globin messenger ribonucleic acid. 112 33

The Friend leukemia virus (FLV)-infected cell line, T-3-Cl-2, undergoes a form of erythroid differentiation in culture when treated with an appropriate inducer, such as dimethylsulfoxide ((CH3)2SO). Thus, whereas untreated cells contain no detectable hemoglobin, treated cells accumulate hemoglobin in quantities comparable to those in the mature mouse red blood cell. We have investigated the mechanism of hemoglobin induction by quantitating the number of globin genes and the amount of globin mRNA in cells before and during the period of hemoglobin accumulation. The results indicate the number of globin genes does not change as the cells accumulate hemogtobin: There are less than 5 globin genes per haploid genome. On the other hand, whereas cells lacking hemoglobin contain little, if any, globin mRNA, hemoglobin-containing cells accumulate, on the average, 8,000 molecules of globin mRNA per cell. The most direct, although, by no means, the only interpretation of these results is that the induction of hemoglobin synthesis involves transcriptional activation of the globin genes. Using this same cell line, we show that mouse globin mRNA sequences are also present in viral particles purified from the culture medium of globin-producing cells. These globin mRNA sequences are absent from viral particles derived from T-3-Cl-2 cells which are not producing globin mRNA. Virus-associated globin mRNA sequences sediment in association with 60S viral RNA complex as well as in free, 9S form. However, under mild denaturing conditions which result in the conversion of viral 60 S RNA to 30S and smaller forms, all the globin sequences sediment as 9S RNA. Appropriate control experiments indicate that the virus-associated globin mRNA is resistant to degradation by exogenous ribonuclease; that exogenously added globin mRNA does not become associated with the 60S viral RNA complex; and that globin mRNA can be detected in virions derived from cells both induced for and constitutively synthesizing globin mRNA. The presence of globin mRNA sequences in FLV particles has important implications in terms of our ability to distinguish between host and viral RNAs in viral particles and in terms of the possible role RNA tumor viruses might play in transduction of genetic information.
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PMID:Induction of globin mRNA in Friend leukemia virus-infected cells and its presence in viral 60S RNA. 117 37

Erythropoiesis in vertebrates is characterized by sequential changes in erythropoietic site, erythroblast morphology, and hemoglobin synthesis. We have examined the expression of globin chains and the major erythroid transcription factor GATA-1 (previously known as GF-1/NF-E1/Eryf 1) from days 7.5 to 17.5 of mouse development. mRNAs for embryonic (epsilon y2, beta H1, and zeta) and adult (alpha and beta) globin chains were quantitated by RNase protection assays. Switching of globins within the alpha-globin cluster (alpha and zeta) was not strictly coordinated with that within the beta-globin cluster (epsilon y2, beta H1, and beta). Regulation of globin switches during development was primarily transcriptional. Of particular note, we found two developmental switches (beta H1 to epsilon y2 and epsilon y2 to beta) in the mouse, more analogous than previously thought to shifts found in human development. The erythroid transcription factor GATA-1, believed to be a principal regulator of genes expressed in erythroid cells, first appeared in the embryo in yolk sac at the time of blood island formation and remained at a low level during embryonic erythropoiesis (8 to 11 days) relative to that found later in fetal liver (12 to 15 days). The rise in GATA-1 mRNA in fetal liver paralleled and preceded the rapid accumulation of adult beta-globin RNA. RNase protection assays and a GATA-1-specific peptide antiserum were used to establish that a single GATA-1 polypeptide is expressed throughout mouse development. Overall, these findings suggest that the levels of this erythroid transcription factor during development may contribute to the differential gene activation characteristic of definitive versus primitive erythropoiesis.
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PMID:Regulated expression of globin chains and the erythroid transcription factor GATA-1 during erythropoiesis in the developing mouse. 170 Oct 19


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