The expression of a plant genome in hnRNA and mRNA.

Representation of genomic kinetic sequence classes and sequence complexities were investigated in nuclear and polysomal RNA of the higher plant Petroselinum sativum (parsley). Two different methods indicated that most if not all polysomal poly(A) -RNA is transcribed from unique sequences. As measured by saturation hybridization in root callus and young leaves 8.7% and 6.2%, respectively, of unique DNA were transcribed in mRNA corresponding to 13.700 and 10.000 average sized genes. Unique nuclear DNA hybridized with an excess of polysomal poly(A)mRNA to the same extent as with total polysomal RNA. 3H-cDNA - poly(A)mRNA hybridization kinetics revealed the presence of two abundance classes with 9.200 and about 30 different mRNAs in leaves and two abundance classes with 10.500 and 960 different mRNAs in callus cells. The existence of plant poly(A)hnRNA was proven both by its fast kinetics of appearance, its length distribution larger than mRNA, and its sequence complexity a few times that of polysomal RNA.     

Characterization of polyadenylated RNA in a protein-producing bacterium, Bacillus brevis 47.

Up to about 50% of the total radioactivity in pulse-labeled RNA in Bacillus brevis 47-5, a high-protein-producing bacterium, was found in the polyadenylated fraction [termed poly(A)-RNA] isolated by adsorption to oligodeoxythymidylic acid-cellulose. Labeled RNA was bound to the cellulose regardless of whether the radioactive precursor was [3H]adenosine or [3H]uridine, showing that the adsorbed material was poly(A)-RNA rather than free poly(A). Poly(A) tracts, isolated after digestion of pulse-labeled RNA with pancreatic and T1 RNases, were homogeneous, with a length of about 95 nucleotides. Susceptibility of the isolated poly(A) tracts to degradation by snake venom phosphodiesterase and polynucleotide phosphorylase indicated that the poly(A) sequences were located directly at the 3'-terminal of the RNA molecules. Comparison of the poly(A)-RNA content in high-protein-producing and nonprotein-producing cells of B. brevis 47 showed much higher levels in the former. Electrophoretic analysis in both denaturing and denaturing polyacrylamide gels of the poly(A)-RNAs showed a heterogeneous population of molecules ranging in size from 23S to 4S. Comparison of the molecular-weight distribution patterns revealed that a significantly greater amount of high-molecular-weight poly(A)-RNA (comigrating with 23S RNA) was present under conditions in which extracellular protein production was high. The possibility that a substantial fraction of the poly(A)-RNA might be involved in the synthesis of extracellular proteins in B. brevis 47 is discussed.     

Sequence complexity of messenger RNA in cotyledons of developing pea (Pisum sativum) seeds

The hybridization kinetics of poly(A)⁺-RNA preparations from the cotyledons of developing pea (Pisum sativum seeds to complementary DNAs have shown that the number of distinct sequences in poly(A)⁺ -RNA decreases from ca 20 000 at the early stage of cotyledon development to ca 200 at a late stage of cotyledon development. The decrease in sequences is accounted for entirely by the disappearance of ‘rare’ poly(A)⁺ -RNAs (< 10³ copies/cell) as seed development proceeds. There is an increase (1–6) in very abundant poly(A)⁺-RNA sequences (? 5 × 10⁵ copies/cell) from early- to mid-developmental stages, concomitantly with the increase in the synthesis of seed-specific storage protein polypeptides. In agreement with the continuing synthesis of most of these polypeptides to the end of seed development, the number of very abundant poly(A)⁺-RNAs is maintained to the late cotyledon development stage. Abundant poly(A)⁺-RNA sequences (ca 10⁴ sequences/cell) increase from 80 to 180 during development, possibly corresponding to the polypeptides which are not storage proteins but are known to be accumulated in pea seeds. Hybridization of single-copy pea genomic DNA sequences to poly(A)⁺-RNA from developing seeds showed that ca 5 % of the single-copy sequences were present in mRNA from mid-development cotyledons. In addition, hybridization of cDNA prepared against poly(A)⁺-RNA from nuclei of early development cotyledons to the corresponding cytoplasmic polysomal poly(A)⁺-RNA showed that the cytoplasmic poly(A)⁺-RNA contained ca 50 % of the sequences present in the nuclei. These results are discussed and interpreted in the light of existing results from similar systems.     

Spironolactone antagonism of aldosterone action on Na+ transport and RNA metabolism in toad bladder epithelium.

In earlier studies, aldosterone increased the incorporation of precursors into a class of cytoplasmic RNA with the characteristics of messenger RNA (mRNA), in toad bladder epithelium. In the present studies, this effect was analyzed further with a competitive antagonist, spironolactone (SC-9420). Paired hemibladders were labeled with 3H-uridine (30 min pulse - 140 min chase), with or without aldosterone (3.5 x 10(-8) M, 7 X 10(-8) M) in the presence or absence of SC-9420 (7 X 10(-6) M, 2.5 X 10(-5) M) at molar ratios of 200:1 to 280:1. Cytoplasmic RNA, either the total phenol-SDS extract or polyadenylated-RNA (poly(A)(+)-RNA) obtained by oligo-deoxythymidylate-cellulose (oligo(dT)-cellulose) chromatography was analyzed in linear 5 -- 20% sucrose gradients. Eight sets of experiments were completed in which the short-circuit current (scc) was monitored for 180 min and the incorporation of 3H-uridine (30 min pulse -- 150 min chase) was simultaneously determined on pools of epithelia from 5 to 10 hemibladders. The fractional change in scc correlated linearly with the fractional change in 3H-uridine of 12S cytoplasmic RNA (r=0.95, p less than 0.001). The poly(A)(+)-RNA fraction had no detectable rRNA or tRNA and gave a heterogeneous pattern, typical of mRNA, in the sucrose gradients. In the presence of exogenous aldosterone, SC-9420 inhibited the incorporation of 3H-uridine into poly(A)(+)-RNA (particularly 12S). These results support the inference that induction of mRNA mediates the action of aldosterone on Na+ transport.     

Messenger RNA during early embryogenesis in Artemia salina: altered translatability and sequence complexity

RNA from developing embryos of Artemia salina (5, 10, and 20 h after re-initiation of development) was translated 3-10 times more efficiently in a rabbit reticulocyte lysate cell-free protein synthesizing system than RNA from dormant gastrulae. The latter did not appear to contain any significant amount of translation inhibitor activity. Ninety percent of the translatable activity in dormant gastrulae was recovered as poly(A)--RNA, whereas 80% of that in post-gastrular developing embryos was present as poly(A)+-RNA. The size of most polypeptides coded for by dormant gastrular RNA was less than 130,000 daltons whereas the size of those coded for by developing embryonic RNA was up to 200,000 daltons, which correlated with a corresponding shift to poly A-containing RNA of higher molecular weight. Two major polypeptides of about 37,000 daltons coded for by dormant gastrular RNA disappeared at 20 h after resumption of development. Hybridization of complementary DNA (cDNA) to a 1000-fold excess of the homologous poly(A)+-RNA revealed the presence of three complexity classes of mRNA. Forty-five percent, 30%, and 25% of RNA in dormant gastrulae were present as high, middle, and low abundance classes comprising about 10, 80, and 9700 species, respectively whereas in the nauplii there were 10, 150, and 7900 species of high, middle, and low abundancy sequences, respectively. Heterologous hybridizations using cDNA complementary to highly abundant messenger population of nauplii (isolated by chromatography on hydroxyapatite) to poly(A)+-RNA from dormant cysts showed considerably divergence in this class of messengers from the two developmental stages. Re-initiation of development of dormant Artemia gastrulae is thus characterized by a "re-programming" seen as a simultaneous and rapid increase in the polyadenylation and translatability of poly(A)+-RNA accompanied by a qualitative change in its sequence complexity.     

mRNA for the plasminogen activator of the urokinase type: translation in oocytes of Xenopus laevis

Poly(A)+-RNA from human kidney and human embryonal lung fibroblasts fractionated by sucrose gradient centrifugation was translated in Xenopus oocytes. Assay for plasminogen-dependent fibrinolytic activity detected synthesis of secreted plasminogen activator and revealed the active fraction of poly(A)+-RNA with a sedimentation coefficient of approximately 23S. Translation products of the active fraction were immunoadsorbed by antiurokinase monoclonal antibodies immobilized on sepharose. Gel electrophoretic analysis of the protein products showed that the 23S fraction of poly(A)+-RNA from human kidney contains mRNA for single-chain urokinase-type plasminogen activator with apparent molecular weight of approximately 50 kDa.     

Messenger ribonucleoprotein complexes of cryptobiotic embryos of Artemia salina.

Poly(A)-containing ribonucleoprotein (poly(A)+-RNP) particles in the post-mitochondrial supernatant of cryptobiotic embryos of Artemia salina were characterized by hybridization to [3H]-poly(U). By sucrose isopycnic centrifugation, approximately 2/3 of poly(A)+-RNPs was found to band at 1.27-1.30 (g/cm3) and the rest 1+/3 at 1.20-1.23 (g/cm3) and below 1.20 (g/cm3). The 1.27-1.30 RNPs could be separated into two density classes, 1.27-1.28 and 1.30 (g/cm3) respectively. The latter RNP class was apparently complexed with ribosomal components because they were completely converted to the former RNP class (free RNPs) by 25 mM EDTA treatment. Further, the 1.30 (g/cm3) RNPs were resolved into several RNP species having sedimentation coefficients above 50 S. which were transformed mostly to 20-30 S rnps in the presence of 25 mM EDTA. The free 20-30 S RNPs contained 8-14 S poly(A)+-RNAs, having the highest template activity in a wheat embryo cell-free system, whereas the 1.20-1.23 poly(A)+-RNPs consisted of 10 S and 16 S RNPs, both of which contained 4 S poly(A)-containing sequences without any template activity.     

Characterization of unstable poly(A)-RNA in Caulobacter crescentus

A significant amount of poly(A)-RNA in Caulobacter crescentus is located on polysomes and the size distribution of this polysomal poly(A)-RNA is small compared to the total pulse-labeled RNA in these cells. These observations suggest that the poly(A)-RNA represents a subset of small messenger RNA molecules. Poly(A)-RNA, and presumably the poly(A) portion of these molecules, is extremely unstable: as assayed by binding to oligo(dT)-cellulose the poly(A)-RNA turns over with a chemical half-life of 15–20 s compared to a half-life of approx. 2 min for total cellular messenger RNA. The presence of adenosine in hydrolysates of poly(A) tracts showed that these sequences are located at the 3′-OH end of the RNA. The ratios of AMP/adenosine in the samples confirmed that the length of the A-tracts is approx. 13–17 nucleotides (Ohta, N., Sanders, M. and Newton, A. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 2343–2346).     

The metabolism of a poly(A) minus mRNA fraction in HeLa cells

About 30% of HeLa cell mRNA lacks poly(A) when labeled in the presence of different rRNA inhibitors. Our method of RNA fractionation precludes contamination of the poly(A)^? mRNA with large amounts of poly(A)⁺ sequences. The poly(A)^? species is associated with polyribosomes, has an average sedimentation value equal to or greater than poly(A)⁺ mRNA, and behaves like the poly(A)⁺ mRNA in its sensitivity to EDTA and puromycin release from polyribosomes. There is very little, if any, hybridization at Rot values characteristic of abundant RNA sequences between the poly(A)^? RNA fractions from total cytoplasm or from polyribosomes and ³H-cDNA made to poly(A)⁺ RNA. This indicates that poly(A)^? mRNA does not arise from poly(A)⁺ mRNA by nonadenylation, deadenylation, or degradation of random abundant mRNA sequences. The rate of accumulation of poly(A)^? mRNA larger than 9S in the cytoplasm parallels the accumulation of poly(A)^? mRNA. The poly(A)^? mRNA is maintained as approximately 30% of the total labeled mRNA in a short (90 min) and in a long (20 hr) time period. These data indicate that poly(A)^? mRNA is not short-lived nuclear or cytoplasmic heterogeneous RNA contamination, and that the half-life of the poly(A)^? mRNA may parallel that of the poly(A)⁺ mRNA. Cordycepin appears to almost completely (95%) inhibit poly(A)⁺ mRNA while only partially (60%) inhibiting the poly(A)^? mRNA. The origin of the cordycepin-insensitive mRNA has not been ascertained.     

Detection of gastrin-specific mRNA using oligodeoxynucleotide probes of defined sequence.

Three oligodeoxynucleotides have been chemically synthesized and used as hybridization probes to detect gastrin-specific mRNA within an heterogeneous population of RNA molecules. Using these oligonucleotides whose nucleotide sequences were deduced from the amino acid sequence of the hormone, 0.2 fmol of gastrin mRNA can be detected/microgram of poly(A)-RNA from hog antrums. The 32P-labeled oligonucleotides hybridize specifically to RNA covalently coupled to DBM-paper (Alwine, J.C., Kemp, D.J., and Stark, G.R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5350-5354). Labeled oligonucleotides also hybridize specifically to RNA separated by gel electrophoresis in the presence of CH3HgOH and covalently coupled to DBM-paper. Using this procedure, we have found that the mRNA coding for gastrin contains about 620 nucleotides, which is in agreement with results obtained using gastrin cDNA to determine mRNA size.     

Purification and properties of biologically active rainbow trout testis protamine mRNA.

At least two classes of protamine mRNA are present in both trout testis polysomal RNA and RNA from the postribosomal supernatant fraction of trout testis hormogenate both of which direct the synthesis of protamine in a Krebs II ascites S-30. One contains poly(A) tracts and the other is devoid of poly(A). Sucrose gradient analyses showed that the poly(A) containing protamine mRNA (poly(A) (+)) sedimented IN THE 6 S region with a shoulder in the 4 S region while the protamine mRNA devoid of poly(A) (poly(A) (-)) appeared to sediment at about 4 S and could not be resolved from tRNA. Analysis of the poly(A) (+) protamine mRNA by boundary sedimentation in an analytical ultracentrifuge showed a sedimentation coefficient of 5.7 S, a value which gives rise to an estimate of 165 to 170 nucleotides per molecule. The poly(A) (+) protamine mRNA migrated as a single species in formamide-containing polyacrylamide gels and its mobility in relation to markers of tRNA (4 S) and 5 S RNA was consistent with its sedimentation velocity of 6 S. The RNA present in the major band on an aqueous polyacrylamide gel was extracted and shown to code for protamine in a wheat germ cell-free system.     

Isolation and characteristics of poly(A+) RNA in the lactating bovine udder

Total cellular poly(A+)-RNA was isolated from a lactating cow mammary gland. The poly(A+)-RNA molecules exhibit a heterogeneous distribution from 500 to 5000 nucleotides (average size--1600 nucleotides) and are made up of three main fractions (1550, 950 and 600 nucleotides) possessing a high template activity during translation in vitro. Optimal conditions for poly(A+)-RNA translation in a cell-free protein-synthesizing system from wheat embryos were elaborated. Immunochemical analysis of translation products revealed that 30% of the synthesized polypeptides are precipitated by immunoglobulins against cow milk proteins. Using hybridization with homologous cDNA, the kinetic complexity and heterogeneity of total cellular poly(A+)-RNA were investigated. This population was shown to consist of four classes differing in the diversity of their nucleotide sequences and the number of copies per cell. The total amount of the poly(A+)-RNA species in the cells of a lactating cow mammary gland is 9200, i.e., 0.46% of the genome complexity.     

Community structure of bacteria and fungi in aerosols of a pig confinement building

Modern intensive husbandry practices can create poor indoor air quality, with high levels of airborne dust, endotoxins, ammonia, and microorganisms. Air in a sow breeding barn was investigated to determine the biomass composition of bioaerosols using molecular methods supplemented with microscopic and cultivation-dependent approaches. A total of 2.7?±?0.7?×?10(7) bacterial cells?m(-3) air and 1.2?±?0.3?×?10(6) fungi spores?m(-3) were detected, corresponding to the fungal biovolume constituted 98% of the total microbial biovolume (fungal and bacterial). Fifty-two percent of all 4',6-diamidino-2-phenyl indole-stained cells were detectable with fluorescence in situ hybridization (FISH) with a general bacterial probe mixture. Quantitative FISH of the bacterial consortium revealed Firmicutes as the dominant group with Streptococcus as the major genus, while Actinobacteria constituted 10% of the detectable bacteria. Additionally, the study revealed an abundant and diverse fungal community including species not previously found in similar environments. The most abundant fungal 18S rRNA gene clone sequences identified affiliated with the Aspergillus-Eurotium cluster, but among others, species of Wallemia, Mucorales, and Russulales were detected. For both fungi and anaerobic bacteria, a hitherto undescribed diversity was found in bioaerosols from a modern sow breeding barn, which potentially could create poor indoor air quality, although their effect on the health of farmworkers and stock still is not resolved.