The putative 15 S precursor of globin mRNA contains a poly (A) sequence

[3H] Uridine or [3H] adenosine pulse-labelled nuclear RNA was isolated from chicken immature red blood cells and separated on denaturing formamide sucrose gradients. RNA of each gradient fraction was hybridized with unlabelled globin DNA complementary to mRNA (cDNA) and subsequently digested by RNAase A and RNAase T1. The experiments revealed two RNA species with globin coding sequences sedimenting 9 S and approx. 15 S, the latter probably representing a precursor of 9 S globin mRNA. A poly (A) sequence was demonstrated in this RNA by two different approaches. Nuclear RNA pulse-labelled with [3H] uridine was fractionated by chromatography on poly (U)-Sepharose. Part of the 15 S precursor was found in the poly(A)-containing RNA. In the second approach 15 S RNA pulse-labelled with [3H]adenosine was hybridized with globin cDNA, incubated with RNAase A and RNAase T1 and subjected to chromatography on hydroxyapatite. The hybrids were isolated and after separation of the strands degraded with DNAase I, RNAase A and RNAase T1. By this procedure poly(A) sequences of approximately 100 nucleotides could be isolated from the 15 S RNA with globin coding sequences. The poly(A) sequence was completely degraded by RNAase T2.     

Determination of polyadenylate-rich ribonucleic acid in the nucleus and in the cytoplasm of plasmacytoma cells.

A number of parameters affecting the adsorption of rRNA and poly(A)-containing RNA to Millipore filters were investigated separately. Binding of both types of RNA to the filter was dependent on the concentration of RNA, pH and Mg2+ concentration of the reaction mixture. Both types of RNA bound to the filter optimally at slightly acid pH values. The binding of poly(A)-containing RNA to the filter exhibited a broad pH-dependence compared with that of rRNA. The ratio of poly(A)-rich RNA/rRNA retained by the filter was maximal between pH7 and 8. The presence of 1 mM-EDTA or a high concentration of NaCl (over 0.5M) decreased the affinity of RNA for the filter. The amount of poly(A)-containing RNA in the nucleus and in the cytoplasm of a plasmacytoma cell line (MPC-11) labelled with [32P]Pi was determined by the Millipore-filter technique under conditions that minimized contamination by rRNA. These data were compared with the estimations made by oligo(dT)-cellulose chromatography. The results obtained by these two methods were in good agreement for RNA labelled for short periods (up to 2h). In long labelling and pulse-chase experiments, however, contamination of the filter by rRNA of increasing specific radioactivity in the cytoplasm gave an erroneous value for poly(A)-containing RNA by the Millipore-filter technique. Determinations made on the nuclear fraction by these two methods did not show significant variation in short- and long-term labelling experiments.     

Isolation of high quality and yield of RNA from Agaricus bisporus with a simple, inexpensive and reliable method

A method for isolating high purity and quantity RNA from Agaricus bisporus which is rich in proteins, carbohydrate, fiber and secondary metabolites, is described. RNA was extracted from mycelium, primordia, sporophores at two development stages and two post-harvest storage stages as well as from pileipellis, inner cap, gill and stipe of the mature sporophore. The A(260)/A(230) and A(260)/A(280) ratios of isolated RNA from fruiting bodies were both ~2 and the yield was about 200 μg/g fresh wt (FW). The yield of RNA from mycelium was approx. 100 μg/g FW. High quality RNA was also extracted from fruiting body tissues of Lentinus edodes, Pleurotus ostreatus, Flammulina velutipes and Pleurotus eryngii with yields from 130 to 225 μg/g FW. RNA extracted from all samples was intact, as demonstrated by gel electrophoresis and was suitable for downstream molecular applications, including RT-PCR and qPCR.     

Translation and characterization of poly(A)-lacking RNA from lupin root nodules

Total polysomal RNA from yellow lupin root nodules was fractionated by double oligo(dT)-cellulose chromatography. Poly(A)-containing and poly(A)-lacking RNA fractions showed considerable messenger activity in wheat germ and rabbit reticulocyte cell-free systems. The sizing of poly(A)-lacking RNA on sucrose-density gradient gives rise to separation of 14S mRNA from 22-24S mRNA species. A single polypeptide with molecular weight of 22,000 was coded for by 14S mRNA, while two polypeptides with an apparent mol. wt. of 90,000 and 87,000 were the main products of 22-24S mRNA fraction. High concentrations of unfractionated poly(A)-lacking RNA as well as the addition of poly(A) led to preferential synthesis of the 22,000 product. Preliminary results suggest the presence of m7GpppX cap structure at 5' terminus of the separated 14S and 22-24S mRNA species. This comes from the competition experiments with m7GMP and m7GTP as well as from the fact that the poly(A)-lacking RNA preparation was susceptible to methylation by methyl-transferase from vaccinia virus (methylated is the 2'-O-nucleotide adjacent to 7-methylguanosine). Digestion by T1 RNAase of methylated poly(A)-lacking RNA produced two short 5'-terminal oligonucleotides 10 and 17 nucleotides in length.     

草莓果实成熟期间RNA代谢的变化

现已证明,鳄梨和番茄等跃变型果实的成熟涉及基因表达的改变和新蛋白质(酶)的合成(Brady 1987)。但是,关于草莓这类非跃变型果实成熟的生化控制机理却很少报道,它们是否也如同跃变型果实那样与新的核酸和蛋白质合成有关?本实验测定了草莓果实成熟期间poly(A)+RNA含量的变化和poly(A)+RNA的体外翻译活性,目的在于探讨草莓果实的成熟机     

Isolation of poly(A)+ RNA by paper affinity chromatography

Poly(A)+ RNA was isolated from in vitro short-term-labeled total cytoplasmic RNA of Ehrlich ascites tumor cells by oligo(dT) cellulose chromatography. This poly(A)+ RNA fraction was compared with a poly(A)+ RNA fraction isolated by a new procedure which involves specific binding of poly(A)+ RNA to messenger affinity paper (mAP) and its release in hot (70 degrees C) water. In typical experiments 10-11 micrograms (2.3%) of poly(A)+ RNA can be retained from 500 micrograms of total cytoplasmic RNA per cm2 of mAP in a quick one-step procedure. The poly(A)+ RNA preparations isolated by the two methods proved to be almost identical with respect to their fraction in total cytoplasmic RNA, specific radioactivities, sucrose gradient profiles, and translation assays. Since the isolation of poly(A)+ RNA by mAP is much less time consuming than that by oligo(dT) column chromatography and since the poly(A)+ RNA can be recovered from mAP in small volumes, which avoids further loss during precipitations, it can be advantageously used for preparative isolation of poly(A)+ RNA.     

Properties of polyadenylate-associated ribonucleic acid from Saccharomyces cerevisiae ascospores.

Bulk ribonucleic acid (RNA) was isolated from mechanically disrupted ascospores of Saccharomyces cerevisiae. After two passes over an oligo (dT10) cellulose column, the portion which bound, called poly(A)(+), was characterized. It is heterodisperse in size with a mean molecular weight of approximately 4 X 10(5), but contains some species as large as 7 X 10(5). The base composition is similar to vegetative poly(A)(+) RNA. The polyadenylate segment is also heterogenous in size, ranging from 90 to 20 bases in length, with a peak at approximately 60 nucleotides in length. Pulse-labeling of asci with [3H-methyl]methionine yields two "caps," 7-methyl guanosine-5'-triphosphoryl-5'-adenosine (or guanosine) identical to that found in vegetative poly(A)(+) RNA. The poly(A)(+) RNA in spores is found in polyribosomes which are, on the average, smaller than vegetative ones. Long-term labeling studies indicate that the fraction of poly(A)(+) RNA in spores is similar to that in vegetative cells.     

RNA synthesis in the early stage of aerobic incubation of potato tuber discs

RNA synthesis of potato tuber discs during the early periodof their aerobic incubation was investigated by feeding thediscs with ³H-uridine. The rate of total RNA synthesis increasedin two steps during the incubation. The increase during thefirst 2 to 3 hr was small, but that after 3 hr was large. Thelabeled RNAs were separated into poly(A) containing RNA [poly(A)(+) RNA] and poly(A) lacking RNA [poly(A) (–) RNA] bythe use of a poly(U)-Sepharose column. Poly(A) (+) RNA was synthesizedeven in the freshly prepared discs which incorporated little¹⁴C-leucine into a protein fraction, and the synthetic rateof poly(A) (+) RNA increased by about 50% during the first 3hr incubation period, then gradually decreased thereafter. Synthesisof poly(A) (–) RNA continued to increase up to 7 hr afterslicing. When the discs were pulse labeled, the proportion ofradioactivity in poly(A) (+) RNA to that in the total RNA wasmaintained at about 50% until about 3 hr after slicing, butit abruptly decreased between 3 and 5 hr to about 35% whichwas maintained up to 9 hr after slicing. (Received October 12, 1977; )     

Structure, translation, and metabolism of the cytoplasmic copia ribonucleic acid of Drosophila melanogaster

We have characterized the copia RNA in the cytoplasm of cultured Drosophila cells. Copia RNA was detected and purified by hybridization to DNA of the plasmid cDm 1142, which contains the copia sequence. A large fraction (2.2%) of the total cytoplasmic poly(A)+ RNA was found to be copia RNA. Cytoplasmic copia RNA displays all the characteristics expected for a messenger RNA. It possesses a poly(A) tract identical in length with that of total poly(A)+ cytoplasmic RNA. It is associated with polysomes and can be released from this association by treatment with EDTA. When purified copia RNA is added to an mRNA-dependent rabbit reticulocyte lysate, three polypeptides of 51000, 33000, and 21000 daltons are seen. We have not determined if these are different polypeptides or if the two smaller polypeptides are fragments of the 51000-dalton polypeptide. The half-life of copia cytoplasmic RNA was determined in pulse--chase experiments to be 9.5 h; this is 1.6 times longer than the half-life of the intermediate decay class of total poly(A)+ cytoplasmic RNA. These properties provide strong evidence that copia RNA functions in vivo as a messenger RNA.     

小鼠肝脏MicroRNA的提取与分离

本文对小鼠肝脏microRNA进行分离制备及相关性功能研究.采用poly(A)加尾方法,分离提取小鼠肝脏中的microRNA;利用T4 RNA连接酶,在microRNA两端加上连接引物,通过RT PCR制备microRNA的cDNA文库;经过对cDNA文库的质粒连接,克隆测序获得microRNA. 结果显示：获得4条有效序列,其中包括2条已知序列miR 122和2条未知小分子RNA序列.经查证, 2条microRNA片段在miRBase库中未有记录,在二级结构分析中可形成茎环结构,符合microRNA的特征. BLAST比对显示： 2个序列位于小鼠载脂蛋白B基因和小鼠28S核糖体RNA上,可能与基因调控有关,其功能有待进一步研究.     

RNA metabolism of murine leukemia virus II. Endogenous virus-specific RNA in the uninfected BALB/c cell line JLS-V9.

Type C virus-specific RNA sequences of BALB/c endogenous virus were detected in JLS-V9 cells (an uninfected BALB/c derived line) by annealing cell RNA with 3-H-labeled virus-specific DNA. Endogenous viruses used in preparing the 3-H-labeled DNA (mostly xenotropic) was prepared from JLS-V9 cells induced to produce virus with iododeoxyuridine. In whole-cell extracts, two virus-specific RNA species, 38S and 27S, were detected. No 60 to 70S virus-specific RNA was found. The same two species of virus-specific RNA were observed in isolated cytoplasmic RNA and in cytoplasmic RNA selected for polyadenylic acid-containing species by binding and elution from oligo(dT) cellulose. Very little, if any, of the virus-specific RNA was active as messenger RNA on polyribosomes. No virus-specific RNA transcribed from genes coding for the BALB/c endogenous N-tropic virus was detected, since 3-H-labeled DNA prepared from endogenous N-tropic virus did not hybridize measurably with JLS-V9 RNA.     

Localization of the yeast RNA polymerase I-specific subunits

The spatial distribution of four subunits specifically associated to the yeast DNA-dependent RNA polymerase I (RNA pol I) was studied by electron microscopy. A structural model of the native enzyme was determined by cryo-electron microscopy from isolated molecules and was compared with the atomic structure of RNA pol II Delta 4/7, which lacks the specific polypeptides. The two models were aligned and a difference map revealed four additional protein densities present in RNA pol I, which were characterized by immunolabelling. A protruding protein density named stalk was found to contain the RNA pol I-specific subunits A43 and A14. The docking with the atomic structure showed that the stalk protruded from the structure at the same site as the C-terminal domain (CTD) of the largest subunit of RNA pol II. Subunit A49 was placed on top of the clamp whereas subunit A34.5 bound at the entrance of the DNA binding cleft, where it could contact the downstream DNA. The location of the RNA pol I-specific subunits is correlated with their biological activity.     

Determination of the poliovirus RNA polymerase error frequency at eight sites in the viral genome.

The poliovirus RNA polymerase error frequency was measured in vivo at eight sites in the poliovirus genome. The frequency at which specific G residues in poliovirion RNA changed to another base during one round of viral RNA replication was determined. Poliovirion RNA uniformly labeled with 32Pi was hybridized to a synthetic DNA oligonucleotide that was complementary to a sequence in the viral genome that contained a single internal G residue. The nonhybridized viral RNA was digested with RNase T1, and the protected RNA oligonucleotide was purified by gel electrophoresis. The base substitution frequency at the internal G residue was measured by finding the fraction of this RNA oligonucleotide that was resistant to RNase T1 digestion. A mean value of 2.0 x 10(-3) +/- 1.2 x 10(-3) was obtained at two sites. A modification of the above procedure involved the use of 5'-end-labeled RNA oligonucleotides. The mean value of the error frequency determined at eight sites in the viral genome by using this technique was 4.1 x 10(-3) +/- 0.6 x 10(-3). Sequencing two of the RNase T1-resistant RNA oligonucleotides confirmed that the internal G was changed to a C, A, or U residue in most of these oligonucleotides. Thus, our results indicated that the polymerase had a high error frequency in vivo and that there was no significant variation in the values determined at the specific sites examined in this study.     

Evidence for differential regulation of two classes of poly(A) RNA inBlastocladiella emersonii zoospores

The poly(A) RNA in zoospores ofBlastocladiella emersonii contains RNA synthesized during the growth phase (GP poly(A) RNA) and late sporulation (LS poly(A) RNA). LS poly(A) RNA synthesized during the final 30 minutes of sporulation is bound exclusively to polyribosomes which comprise approximately 50% of the total zoospore ribosome population. In contrast, GP poly(A) RNA is bound to zoospore monoribosomes. During the final 30 minutes of sporulation, GP poly(A) RNA which is bound to polyribosomes makes a transition to monoribosomes. Zoospore monoribosomes and RNA extracted from zoospore monoribosomes are inactivein vitro while both zoospore polyribosomes and RNA extracted from zoospore polyribosomes stimulate protein synthesis in the wheat germin vitro system. Sedimentation of poly(A) RNA from zoospore monoribosomes on dimethyl sulfoxide gradients revealed that the GP poly(A) RNA was of sufficiently high molecular weight to code for average-sized proteins. These denaturing gradients failed to activate the zoospore monoribosome RNA. The results suggest that the inability to translate zoospore monoribosomesin vitro is due to some property or modification of the zoospore monoribosome poly(A) RNA. Zoospore monoribosomes bound to poly(A) RNA contain an average of two tRNA molecules while zoospore polyribosomes have an average of less than one tRNA bound. This suggests the two classes of ribosomes are blocked at different steps in the elongation process.     

The synthesis of polyadenylic acid-containing ribonucleic acid by isolated mitochondria from Ehrlich ascites cells.

The synthesis of poly(A)-containing RNA by isolated mitochondria from Ehrlich ascites cells was studied. Isolated mitochondria incorporate [3H]AMP or [3H]UTP into an RNA species that adsorbs on oligo (dT)-cellulose columns or Millipore filters. Hydrolysis of the poly(A)-containing RNA with pancreatic and T1 ribonucleases released a poly(A) sequence that had an electrophoretic mobility slightly faster than 4SE. In comparison, ascites-cell cytosolic poly(A)-containing RNA had a poly(A) tail that had an electrophoretic mobility of about 7SE. Sensitivity of the incorporation of [3H]AMP into poly(A)-containing RNA to ethidium bromide and to atractyloside and lack of sensitivity to immobilized ribonuclease added to the mitochondria after incubation indicated that the site of incorporation was mitochondrial. The poly(A)-containing RNA sedimented with a peak of about 18S, with much material of higher s value. After denaturation at 70 degrees C for 5 min the poly(A)-containing RNA separated into two components of 12S and 16S on a 5-20% (w/v) sucrose density gradient at 4 degrees C, or at 4 degrees and 25 degrees C in the presence of formaldehyde. Poly(A)-containing RNA synthesized in the presence of ethidium bromide sedimented at 5-10S in a 15-33% (w/v) sucrose density gradient at 24 degrees C. The poly(A) tail of this RNA was smaller than that synthesized in the absence of ethidium bromide. The size of the poly(A)-containing RNA (approx. 1300 nucleotides) is about the length necessary for that of mRNA species for the products of mitochondrial protein synthesis observed by ourselves and others.     

Analysis of the membrane-associated poly(A)+RNA in the cytoplasm of dormant Artemia cysts by DNA excess hybridization. Evidence for a nuclear origin

Encysted embryos of Artemia contain latent mRNA, to a large extent associated with a fraction of cytoplasmic membranes. The membranes, purified by EDTA treatment and banding in a sucrose gradient at 1.17 g/cm3, include endoplasmic vesicles and mitochondria. The origin of the membrane-associated poly(A)+RNA was therefore investigated. In gel electrophoresis poly(A)+RNA from the purified membranes of dormant cysts forms two distinct bands at approx. 3 . 10(5) and 5 . 10(5) Da. Later during development the lighter component decreases. Nuclei from dormant cysts are devoid of poly(A)+RNA, while nuclei from developing embryos (50% emergence) contain a predominant poly(A)+RNA component of approx. 5 . 10(5) Da. 125I-labelled preparations of nuclear DNA and of nuclear and membrane-associated poly(A)+RNA were used in reassociation and hybridization experiments with excess nuclear DNA. Poly(A)+RNA from the membranes of dormant cysts hybridized to nuclear DNA to the same extent as the nuclear poly(A)+RNA from developing embryos. The hybridization of labelled, nuclear poly(A)+RNA to nuclear DNA was strongly inhibited by unlabelled membrane RNA from either dormant cysts or developing embryos. It is concluded that the stored, membrane-associated poly(A)+RNA in dormant cysts is essentially of nuclear origin. The 5 . 10(5)-Da component is largely homologous with the corresponding component of nuclear poly(A)+RNA at later stages.