Control of breakdown of the polyadenylate sequence in mammalian polyribosomes: role of poly(adenylic acid)-protein interactions

The poly(adenylic acid) [poly (A)] segment in mouse sarcoma polysomes in not hydrolyzed by snake venom exonuclease under conditions which cause extensive degradation of the poly(A) in deproteinized polysomal RNA. The protecting effect of polysomes is presumably caused by the interaction between the poly(A) sequence and the protein known to be associated with it. This protection is reduced at low KCl concentration, but addition of exogenous RNA restores the protecting effect. The poly(A) segment also becomes susceptible to exonuclease after fragmentation of the polysomes by mild ribonuclease treatment. The latter treatment releases the poly(A) in association with protein. The poly(A) sequence in polysomes in readily degraded by a cytoplasmic extract of S-180 cells. Partial purification leads to a preparation active against the poly(A) in polysomes under conditions where no fragmentation of the messenger RNA is observed. Snake venom exonuclease increases the activity of the cytoplasmic preparation against poly(A) in polysomes. The active cytoplasmic factor appears to interfere with the poly(A)-protein interaction, thus rendering the polynucleotide susceptible to degradation by exonuclease. The poly(A) sequences in polysomes and in free cytoplasmic nucleoprotein particles are hydrolyzed to the same extent. The results suggest that the poly(A) sequence is normally protected from nucleases by virtue of its association with protein. The slow reduction in poly(A) size in cytoplasmic mRNA can be accounted for by a factor capable of interfering with the poly(A)-protein interaction. The latter interaction seems also dependent on the structural integrity of the polysomes or messenger ribonucleoproteins. It is suggested that a polynucleotide segment adjacent to the poly(A) can modulate the affinity of the protein for the latter sequence, thus permitting control of poly(A) stability in individual messenger RNAs.     

A procedure for the quantitative recovery of homogeneous populations of undegraded free and bound polysomes from rat liver.

A procedure is described for the preparation of free and bound polysomes from whole homogenates of rat liver tissue. Liver is homogenized in a conventional medium containing glutathione; then after a 12-min centrifugation at 131000g, the free polysomes in the supernatant are saved, while the membrane-bound polysomes in the pellet are suspended in a mixture of ribonuclease inhibitors (cell sap, 250 mM KCl, and glutathione), homogenized in the presence of detergent (Triton X-100), centirfuged for 5 min at 1470g, decanted, and treated with deoxycholate; the polysomes in the two supernatants are harvested by centrifugation through sucrose gradients containing 250 mM KCl and cell sap. Free and bound polysomes prepared in this manner are undegraded, equally active in cell-free protein synthesis, and virtually free of ribonuclease, membranous material, glycogen, deoxycholate, completed protein, and cross-contamination. The recovery of polysomes is approximately 95% and the distribution between the free and membrane-bound state is 25 and 75%, respectively. The molecular weight profiles after sodium dodecyl sulfate-acrylamide gel electrophoresis of the polypeptides completed and released by free and bound polysomes in vitro are different, indicating that there are quantitative differences in the synthesis of various size polypeptides between the two polysome classes. The differential centrifugation procedure is rapid and reproducible, requires much less ultracentrifugation than the isopycnic technique, and provides a nearly quantitative means of separating free and bound polysomes.     

Proteins of polyribosome-bound informosome of germinating wheat embryos

Proteins of polyribosome-bound informosomes of germinating wheat embryos were studied by electrophoresis in polyacrylamide gel in presence of sodium dodecyl sulfate. liberation of informosomal proteins was achieved by mild ribonuclease treatment of polyribosomes. It was shown, that proteins of informosomes associated with polyribosomes contain polypeptides with molecular weights of 86 000, 75 000, 72 000, 66 000, 52 000 and 34 000. The milecular weights of two most prominent proteins were 86 000 and 52 000. The treatment of polyribosomes with 0.5 M KCl resulted in the loss of large part of informosomal proteins, which are revealed in the KCl-wash.     

Isolation and characterization of poly(adenylic acid)-containing messenger ribonucleic acid from rat liver polysomes

Undegraded rat liver polysomes were obtained after homogenizing the tissue in a medium containing NH4Cl, heparine, and yeast tRNA. Purification of poly(A)-containing RNA from polysomal RNA was accomplished by affinity chromatography on oligo(dT)-cellulose columns. Poly(A)-containing RNA molecules were monitored by the formation of ribonuclease-resistant hybrids with [3H]poly(U). To improve the separation of messenger RNA and ribosomal RNA by oligo(dT)-cellulose it was found essential to dissociate the aggregates formed between both molecular species by heat treatment in the presence of dimethylsulfoxide (Me2SO) prior to chromatography. Sucrose gradient analysis under denaturing conditions showed that the preparations obtained were virtually free of ribosomal RNA. Poly(A)-containing RNA constituted approx. 2.2% of the total polysomal RNA and the number average size was 1500--1800 nucleotides, as judged by sedimentation analysis on sucrose density gradients containing Me2SO. Approximately 8.2% of the purified preparation obtained was able to anneal with [3H]poly(U); the number average nucleotide length of the poly(A) segment of the RNA population was calculated to be 133 adenylate residues. Based on these values, our preparations appear to be greater than 90% pure. The RNA fractions obtained after oligo(dT)-cellulose chromatography were used to direct the synthesis of liver polypeptides in a heterologous cell-free system derived from wheat-germ. The system was optimized with respect to monovalent and divalent cations, and presence of polyamines (spermine). More than 65% of the translational activity present in the unfractionated polysomal RNA was recovered in the final poly(A)-containing RNA fraction. However, about 25% of the activity was found to be associated with the unbound fraction which was essentially free of poly(A)-containing RNA. Immunoprecipitation analysis with a specific antiserum to rat serum albumin demonstrated that about 6--8% of the labeled synthetic products translated from the poly(A)-containing RNA sample corresponded to serum albumin. Analysis of the translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a heterogeneous distribution of molecular sizes ranging from 15 000 to greater than 70 000 daltons. Spermine not only increased the overall yield and extent of protein synthesis, but also resulted in higher yields of large protein products. Under optimal translation conditions a discrete peak representing about 7% of the total radioactivity was observed to migrate with rat serum albumin.     

Stored Messenger Ribonucleoprotein Particles in Differentiated Sclerotia of Physarum polycephalum

Starvation induces vegetative microplasmodia of Physarum polycephalum to differentiate into translationally-dormant sclerotia. The existence and the biochemical nature of stored mRNA in sclerotia is examined in this report. The sclerotia contain about 50% of the poly(A)-containing RNA [poly(A)+RNA] complement of microplasmodia as determined by [³H]-poly(U) hybridization. The sclerotial poly(A)+RNA sequences are associated with proteins in a ribonucleoprotein complex [poly(A)+mRNP] which sediments more slowly than the polysomes. Sclerotial poly(A)+RNP sediments more rapidly than poly(A)+RNP derived from the polysomes of microplasmodia despite the occurrence of poly(A)+RNA molecules of a similar size in both particles suggesting the existence of differences in protein composition. Isolation of poly(A)+RNP by oligo (dT)-cellulose chromatography and the analysis of its associated proteins by polyacrylamide gel electrophoresis show that sclerotial poly(A)+RNP contains at least 14 major polypeptides, 11 of which are different in electrophoretic mobility from the polypeptides found in polysomal poly(A)+RNP. Three of the sclerotial poly(A)+RNP polypeptides are associated with the poly(A) sequence (18, 46, and 52 × 10³ mol. wt. components), while the remaining eight are presumably bound to non-poly(A) portions of the poly(A)+RNA. Although distinct from polysomal poly(A)+RNP, the sclerotial poly(A)+RNP is similar in sedimentation behavior and protein composition (with two exceptions) to the microplasmodial free cytoplasmic poly(A)+RNP. The results suggest that dormant sclerotia store mRNA sequences in association with a distinct set of proteins and that these proteins are similar to those associated with the free cytoplasmic poly(A)+RNP of vegetative plasmodia.     

Association of messenger ribonucleic acid with mammalian microsomal membranes: characterization by analysis of cell-free translation products.

Total rough microsomes, isolated from the dog pancreas, were stripped of membranes-bound polysomes by treatment with either EDTA or puromycin and 0.5 M KCl. The stripped microsomal membranes were isolated relatively free from contamination, by using buoyant density centrifugation, and mRNA was isolated from both the membrane fraction and the released material. Depending on the method used to strip the rough microsomes, we found a variable but small percentage (3--15%) of the cellular poly(A)-containing mRNA attached to the microsomal membranes. Reextraction of isolated microsomal membranes with puromycin and 0.5 M KCl reduced the content of membrane-associated mRNA by approximately 50%, resulting in less than 2% of the total membrane-bound polysomal mRNA remaining associated with the microsomal membranes. The membrane-associated mRNA was characterized by translation in the wheat germ cell-free protein synthesizing system, and the products were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The translation products of the membrane-associated mRNA were identical with those from the total pancreas mRNA and also with those obtained by using mRNA isolated from material released directly from the rough microsomes.     

Cell-free synthesis of a larger-molecular-weight precursor of cytochrome c oxidase subunit V from rat liver and the distribution of its mRNA between free and membrane-bound polysomes

Poly(A)-rich RNA from phenol-extracted rat liver polysomes was translated in a heterologous cell-free system derived from wheat germs. The labeled translation products were incubated with an antiserum against cytochrome c oxidase subunit V. After immunoprecipitation and affinity chromatography with protein-A-Sepharose, the isolated antigen-immunoglobulin complexes were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. Only one protein with an apparent molecular weight of 15 500 was visualized. In immunocompetition experiments with unlabeled individual cytochrome c oxidase subunits IV, V, VI or VII only subunit V could compete with the 15 500-Mr protein synthesized in vitro. Two-dimensional fingerprints of cytochrome c oxidase subunit V and the polypeptide synthesized in vitro showed a high degree of similarity. It is concluded that the cytochrome c oxidase subunit V is synthesized as a precursor with an amino-terminal extension of about 25 amino acids. It was possible to convert the precursor of cytochrome c oxidase subunit V synthesized in vitro to its mature form by intact mitochondria as well as by submitochondrial particles. A chain length of 830 +/- 70 nucleotides was estimated for the poly(A)-rich mRNA of the higher-molecular-weight precursor of rat liver cytochrome c oxidase subunit V. Assuming a molecular weight of 15 500 for the precursor a non-coding region of about 300 nucleotides must exist. In experiments on the site of synthesis it is shown that the poly(A)-rich RNA for the higher-molecular-weight precursor of cytochrome c oxidase subunit V is found in free, loosely and tightly membrane-bound polyribosomes.     

Metabolism of Poly(A) in Plant Cells: Discrete Classes Associated with Free and Membrane-bound Polysomes

In the subapical region of dark-grown pea epicotyls about 40% of the total polysomes are associated with membranes. The presence of poly(A) in polysomal mRNA was detected by hybridization of unlabeled RNA with (3)H-poly(U). Both free mRNA and messenger ribonucleoprotein particles in polysomes hybridize with (3)H-poly(U) quantitatively. The binding of (3)H-poly(U) to polysomes is increased by treatment with the detergent sodium dodecyl sulfate. Since detergent influenced the (3)H-poly(U) binding more in membrane-bound polysomes than in free, there may be more protein(s) associated with the poly(A) portion of the mRNA in membrane-bound polysomes. Analysis of the poly(A) segments isolated from the mRNA of these two classes of polysomes indicates that there are discrete classes of poly(A) and they appear to be differentially associated with free and membrane-bound polysomes. Mean size distribution of poly(A) in free polysomes is larger than in membrane-bound polysomes.Following treatment (2 days) with the plant growth hormone indoleacetic acid, there is a gradual decrease in the mean length of total poly(A), which appears to correspond to a decrease in the size of the polysomes and their associated mRNA.     

Polypeptides of a Light-Harvesting Complex of the Diatom Phaeodactylum tricornutum Are Synthesized in the Cytoplasm of the Cell as Precursors

A light-harvesting fucoxanthin-chlorophyll a/c-protein complex has been isolated from the diatom Phaeodactylum tricornutum by detergent extraction of thylakoid membranes coupled with sucrose density gradient centrifugation. The isolated complex was devoid of photochemical activity and displayed spectral characteristics consistent with light harvesting function. It has three major polypeptides of apparent molecular weights 18,000, 19,000, and 19,500 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Using protein synthesis inhibitors, these polypeptides were shown to be synthesized on 80S cytoplasmic ribosomes. Antibodies raised to a mixture of the 19,000 and 19,500 dalton components of the complex were used to demonstrate structural similarity among the three polypeptide components. Immunoprecipitation from primary translation products synthesized in a reticulocyte lysate system primed with P. tricornutum poly(A) RNA, indicates that the polypeptide components are synthesized as precursors 3,000 to 5,000 daltons larger than the mature polypeptides.     

Purification and characterization of a ribonuclease specific for poly(U) and poly(C) from the larvae of Ceratitis capitata

A specific ribonuclease was detected and purified to homogeneity from six-day-old larvae of the insect Ceratitis capitata and its homogeneity was checked by analysis in polyacrylamide gels in the presence of sodium dodecyl sulfate. The nuclease specifically degrades poly(U) and poly(C) whilst it fails to do so with other single-stranded homopolyribonucleotides. The enzyme has a pH optimum in the region 7-9 and relative molecular mass of about 25,000. The effect of this ribonuclease on the integrity of RNAs isolated from six-day-old larvae or rat liver was also studied.     

The cytoplasmic distribution and characterization of poly(A)+RNA in oocytes and embryos of Drosophilia.

Using the presence of poly(A) tracts as a marker for mRNA, we have examined the distribution of this class of RNA between polysomes and free RNP particles. This has been done in mature oocytes and in embryos aged for various times from fertilization through to hatching of a larva. The proportion of ribosomes that are in polysomes to those that are not has been calculated. In mature oocytes, 58% of the poly(A)⁺ RNA and 72% of the ribosomes are not in polysomes. By 1 hr, this drops to 51% of the poly(A)⁺ RNA and 48% of the ribosomes. By 7 hr, a plateau is reached: 30% of each are not in polysomes. The poly(A)⁺ RNA in the cytoplasm of oocytes and 1-hr embryos is found in particles with an average size of 50S and a range of 30–70S. The poly(A)⁺ RNA ranges in size from 7 to 40S, with an average size of 22S. The polyA from this RNA is 50–200 nucleotides long with an average of 115 nucleotides. These data have allowed us to calculate that 1–2% of the total RNA is poly(A)⁺ RNA.     

Solubilization and reconstruction of microsomal AMP-deaminase from skeletal muscles]

Microsomal AMP-deaminase was solubilized by 0.5 M KCl after treatment of microsomal membranes with 0.12 M KCl. Using disc-electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate one major protein component (mol. weight about 90 000) and three minor ones with molecular weights of 110 000, 80 000, and 60 000 were found in the soluble fraction. In addition to proteins, the fraction was found in the soluble fraction. In addition to proteins, the fraction was found to contain a small amount of phospholipids. The deaminase found in the solution may be reconstructed into the membranes at a decrease in KCl concentration, part of enzyme being bound in the inactive form under excess of the soluble fraction. Deaminase binding to the membranes is unaffected by the changes within the pH range of 6.2--7.8 and temperature range of 4--10 degrees C. It is assumed that AMP-deaminase is bound to other membrane components by electrostatic bonds.     

Isolation of rat liver albumin messenger RNA.

Rat liver albumin messenger RNA has been purified to apparent homogeneity by means of polysome immunoprecipitation and poly(U)-Sepharose affinity chromatography. Specific polysomes synthesizing albumin were separated from total liver polysomes through a double antibody technique which allowed isolation of a specific immunoprecipitate. The albumin-polysome immunoprecipitate was dissolved in detergent and the polysomal RNA was separated from protein by sucrose gradient centrifugation. Albumin mRNA was then separated from ribosomal RNA by affinity chromatography through the binding of poly(U)-Sepharose to the polyadenylate 3' terminus of the mRNA. Pure albumin mRNA migrated as an 18 S peak on 85% formamide-containing linear sucrose gradients and as a 22 S peak on 2.5% polyacrylamide gels in sodium dodecyl sulfate. It coded for the translation of authentic liver albumin when added to a heterologous protein-synthesizing cell-free system derived from either rabbit reticulocyte lysates or wheat germ extracts. Translation analysis in reticulocyte lysates indicated that albumin polysomes were purified approximately 9-fold from total liver polysomes, and that albumin mRNA was purified approximately 74-fold from albumin polysomal RNA. The total translation product in the mRNA-dependent wheat germ system, upon addition of the pure mRNA, was identified as authentic albumin by means of gel electrophoresis and tryptic peptide chromatography.     

Multiple oligo(A) tracts associated with inactive sea urchin maternal mRNA sequences

Maternal RNA of sea urchin eggs and embryos was analyzed for short poly(A) sequences by digesting hybrids formed between [³H]poly(U) and poly(A) with RNase at 4°C. When the undigested [³H]poly(U) is precipitated with CTAB, all (A)_n tracts longer than 6 nucleotides are detected. This assay revealed a poly(A) content severalfold higher than is obtained with a similar assay using RNase at higher temperatures. On polyacrylamide gel electrophoresis, most of the previously undetected (A)_n tracts ran as a peak of oligo(A) of less than 20 nucleotides which accumulated at the dye front. The oligo(A) sequences were resolved into a single peak of (A)₁₀ when sized on Sephadex G100. These (A)₁₀ sequences were associated with large mRNA-sized molecules of about 3000 nucloetides average length which comprised 0.5 to 2% of the total maternal RNA. However, the (A)₁₀ sequences were not in mRNA molecules containing 3′-terminal poly(A) of 50–120 nucleotides nor did they remain in RNA that entered polysomes upon fertilization. However, hybridization studies showed that all sequences represented in the maternal poly(A)-containing RNA appeared to be present in the RNA molecules containing only (A)₁₀ sequences. The results suggest that the (A)₁₀-containing RNA might be incompletely processed mRNA precursor-like molecules.     

Organization of vitellogenin polysomes, size of the mRNA and polyadenylate fragment.

A heavy polysome fraction containing vitellogenin mRNA was isolated from the liver of oestradiol-treated chicks. As determined by urea-polyacrylamide gel electrophoresis, the molecular weight of vitellogenin mRNA is about 2.5 x 10(6). The mRNA contains a polyadenylate segment of about 220 nucleotides at the 3' end. The remaining 7000 nucleotides are sufficient to code for a polypeptide of Mr about 270000. Combining 'run off' experiments of heavy polysomes in vitro together with radioimmunoprecipitation and polyacrylamide gel electrophoresis of the translation product, we concluded that vitellogenin mRNA is probably monocistronic and the 2.5 x 10(6)-Mr mRNA codes for two polypeptides, Mr 30000 and 240000. The largest polypeptide is, in our cell-free system and liver homogenate, readily cleaved into smaller peptides.     

Storage Protein Synthesis in Maize: Isolation of Zein-synthesizing Polyribosomes

Undegraded free and membrane-bound polysomes were isolated from developing kernels of Zea mays L. frozen in liquid nitrogen. Freezing in liquid nitrogen was a prerequisite for preserving polysome structure in stored kernels. Membrane-bound polysomes from 22-day post-pollination kernels ground in high pH buffers containing 50 mm Mg(2+) contained unique classes of large polysomes. These large polysomes were sensitive to ribonuclease, and electron micrographs verified that they were not formed by aggregation. The membrane-bound polysomes were the principal site of zein synthesis, since the major protein synthesized in vitro was similar to purified zein in its ethanol solubility and mobility on sodium dodecyl sulfate polyacrylamide gels.     

Comparison of polypeptides from cultured human fibroblasts and sarcoma cells.

The proteins in cell layers of cultured normal diploid human skin (ES, ER) and lung (WI-38) fibroblasts were compared to those of SV40-transformed human fibroblasts (WI-38/VA-13), human rhabdomyosarcoma (RD) and fibrosarcoma (HT-1080) cells using metabolic amino acid and sugar labeling and surface labeling with tritiated sodium borohydride after oxidation with galactose oxidase. The labeled proteins were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography (fluorography). A transformation-associated decrease in the pericellular glycoprotein fibronectin (subunit molecular weight, 220 000) and in the synthesis of a set of polypeptides in the 130 000--180 000 dalton region was seen. Synthesis of a glycosylated 160 000 dalton polypeptide was markedly reduced. In transformed cells distinct increases of several specific polypeptides was detected in both [35S]methionine and [3H] mannose incorporation experiments but not using the surface labeling method.     

Isolation and partial purification of ceruloplasmin messenger RNA from rat liver

Partially purified ceruloplasmin mRNA was isolated using indirect immunoprecipitation of rat liver polysomes and poly(U)-Sepharose chromatography of polysomal RNA. This RNA programmed the synthesis of ceruloplasmin polypeptides in a cell-free system from mitochondria. Immunochemical analysis of the translation products revealed a 40-fold enrichment of the ceruloplasmin mRNA activity. The purified ceruloplasmin mRNA migrated as a major homogeneous component with an apparent molecular weight about 1×10⁶ daltons in polyacrylamide gels containing sodium dodecyl sulfate. The immunoprecipitated products of the cell-free translation had molecular weights in the range 4.5–5.4×10⁴ daltons as estimated by gel-electrophoresis under denaturating conditions. These values approach the weight of the half-molecule of native ceruloplasmin.     

Studies on a nucleoprotein prepared from rat liver polysomes by digestion with T1 ribonuclease

1. Treatment of rat liver polysomes in a buffer containing 2.5mm-magnesium chloride with T(1) ribonuclease at a concentration of 330units/ml. of reaction medium at 37 degrees for 2hr. leads to the production of an insoluble nucleoprotein. 2. On the bases of analysis for protein and RNA and of u.v.-absorption spectra the nucleoprotein appears to have lost approx. 60% of the structural RNA originally present in the ribosome. Degradation of (3)H-labelled polysomes (structural RNA labelled with orotic acid) with T(1) ribonuclease leads to nucleoprotein preparations retaining approx. 30% of the radioactivity originally present in the polysomes. By means of sucrose-density-gradient centrifugation it is shown that the nucleoprotein preparations are free of single 73s ribosomes and ribosomal subunits. No evidence for the presence of 28s and 18s structural RNA was obtained on examination of extracted nucleoprotein-particle RNA by means of sucrose-density-gradient centrifugation. 3. Digestion of washed polysomes carrying (14)C-labelled nascent peptide chains with T(1) ribonuclease gives a nucleoprotein particle that retains approx. 70% of the original labelled chains. Treatment of labelled nucleoprotein particles with 1mm-puromycin in the absence of transfer factors releases 20% of the labelled chains. Addition of GTP (0.48mumole) increases this release to 37%. 4. Treatment of nucleoprotein particles carrying (14)C-labelled peptide chains with either EDTA (50mm) or ammonium chloride (0.5m) brings about a small release of labelled material (approx. 15%). 5. Disruption of nucleoprotein particles carrying (14)C-labelled peptide chains with either sodium dodecyl sulphate or 2m-lithium chloride, followed by addition of transfer RNA as marker and chromatography on Sephadex G-200, show in both cases that considerable amounts of labelled peptide material move well ahead of the added transfer RNA marker. Further, if nucleoprotein particles carrying labelled peptide chains are treated with 0.3m-potassium hydroxide at 20 degrees for 24 hr., neutralized to pH7.6, and then chromatographed on Sephadex G-200, the labelled peptide material moves much closer to the added transfer RNA marker. These results suggest that a proportion of the nascent (14)C-labelled peptides on the nucleoprotein are attached to transfer RNA or large fragments of transfer RNA. 6. [(3)H]Polyuridylic acid binds to nucleoprotein particles in 1mm-magnesium chloride. The rate of binding is rapid when measured at 20 degrees .     

Purification of messenger RNA coding for glycine methyltransferase from rat liver

Rat liver messenger RNA coding for glycine methyltransferase was associated preferentially with free polysomes. The mRNA was purified about 1000-fold over the total poly(A)-containing RNA by specific immunoadsorption of polysomes to protein A-Sepharose followed by oligo(dT)-cellulose column chromatography. Sodium dodecyl sulfate-gel electrophoresis of the in vitro translation products in a rabbit reticulocyte lysate system revealed only one major band which migrated to the position of the purified glycine methyltransferase subunit. The result shows that the mRNA isolated is nearly homogeneous and suggests that no precursor form of the enzyme existed. The mRNA sedimented at the position slightly smaller than 18 S rRNA in a sucrose density-gradient centrifugation and was shown to contain about 1,300 nucleotides by the Northern blot hybridization analysis with a cDNA probe.