Trans-species small RNAs move long distances in a parasitic plant complex

Parasitic plants exchange various types of RNAs with their host plants, including mRNA, and small non-coding RNA. Among small non-coding RNAs, miRNA production is known to be induced at the haustorial interface. The induced miRNAs transfer to the host plant and activate secondary siRNA production to silence target genes in the host. In addition to interfacial transfer, long-distance movement of the small RNAs has also been known to mediate signaling and regulate biological processes. In this study, we tested the long-distance movement of trans-species small RNAs in a parasitic-plant complex. Small RNA-Seq was performed using a complex of a stem parasitic plant, Cuscuta campestris, and a host, Arabidopsis thaliana. In the host plant’s parasitized stem, genes involved in the production of secondary siRNA, AtSGS3 and AtRDR6, were upregulated, and 22-nt small RNA was enriched concomitantly, suggesting the activation of secondary siRNA production. Stem-loop RT-PCR and subsequent sequencing experimentally confirmed the mobility of the small RNAs. Trans-species mobile small RNAs were detected in the parasitic interface and also in distant organs. To clarify the mode of long-distance translocation, we examined whether C. campestris-derived small RNA moves long distances in A. thaliana sgs3 and rdr6 mutants or not. Mobility of C. campestris-derived small RNA in sgs3 and rdr6 mutants suggested the occurrence of direct long-distance transport without secondary siRNA production in the recipient plant.     

Structural features unique to the 5 S ribosomal RNAs of the thermophilic cyanobacterium Synechococcus lividus III and the green plant chloroplasts

The complete nucleotide sequence of the 5 S ribosomal RNA from the thermophilic cyanobacterium Synechococcus lividus III was determined. The sequence is: ^5′U-C- C-U-G-G-U-G-G-U-G-A-U-G-G-C-G-A-U-G-U-G-G-A-C-C-C-A-C-A-C-U-C-A-U-C- C-A-U-C-C-C-G-A-A-C-U-G-A-G-U-G-G-U-G-A-A-A-C-G-C-A-U-U-U-G-C-G-G-C- G-A-C-G-A-U-A-G-U-U-G-G-A-G-G-G-U-A-G-C-C-U-C-C-U-G-U-C-A-A-A-A-U-A- G-C-U-A-A-C-C-G-C-C-A-G-G-G-U_OH^3′This 5 S RNA has regional structural characteristics that are found in the green plant chloroplast 5 S RNAs and not in other known sequences of 5 S ribosomal RNAs. These homologies suggest a close phylogenetic relationship between S. lividus and the green plant chloroplasts.     

Changes in Gene Expression during Tomato Fruit Ripening

Total proteins from pericarp tissue of different chronological ages from normally ripening tomato (Lycopersicon esculentum Mill. cv Rutgers) fruits and from fruits of the isogenic ripening-impaired mutants rin, nor, and Nr were extracted and separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Analysis of the stained bands revealed increases in 5 polypeptides (94, 44, 34, 20, and 12 kilodaltons), decreases in 12 polypeptides (106, 98, 88, 76, 64, 52, 48, 45, 36, 28, 25, and 15 kilodaltons), and fluctuations in 5 polypeptides (85, 60, 26, 21, and 16 kilodaltons) as normal ripening proceeded. Several polypeptides present in ripening normal pericarp exhibited very low or undetectable levels in developing mutant pericarp. Total RNAs extracted from various stages of Rutgers pericarp and from 60 to 65 days old rin, nor, and Nr pericarp were fractionated into poly(A)⁺ and poly(A)⁻ RNAs. Peak levels of total RNA, poly(A)⁺ RNA, and poly(A)⁺ RNA as percent of total RNA occurred between the mature green to breaker stages of normal pericarp. In vitro translation of poly(A)⁺ RNAs from normal pericarp in rabbit reticulocyte lysates revealed increases in mRNAs for 9 polypeptides (116, 89, 70, 42, 38, 33, 31, 29, and 26 kilodaltons), decreases in mRNAs for 2 polypeptides (41 and 35 kilodaltons), and fluctuations in mRNAs for 5 polypeptides (156, 53, 39, 30, and 14 kilodaltons) during normal ripening. Analysis of two-dimensional separation of in vitro translated polypeptides from poly(A)⁺ RNAs isolated from different developmental stages revealed even more extensive changes in mRNA populations during ripening. In addition, a polygalacturonase precursor (54 kilodaltons) was immunoprecipitated from breaker, turning, red ripe, and 65 days old Nr in vitro translation products.     

The psu1+ amber suppressor gene of bacteriophage T4: identification of its amino acid and transfer RNA

Previous work identified the psu⁺₁ amber suppressor gene of bacteriophage T4. Analysis of protein arising from suppression now shows that psu⁺₁ specifies the insertion of serine in response to the amber triplet. The efficiency of suppression is 70%.The psu₁⁺ gene affects a serine transfer RNA coded by bacteriophage T4. Comparative ribonuclease T₁ fingerprint analysis of the serine transfer RNAs made by wild type T4 and psu⁺₁ strains shows a specific alteration in the patterns, presumably reflecting a mutational alteration in the anticodon of the transfer RNA. Mutations which result in the loss of suppressor activity define two genes: one is apparently the structural gene for the serine transfer RNA; the function of the second gene, M1, is less clear. Mutational inactivation of either gene prevents the appearance of the serine transfer RNA and a second transfer RNA, which has not yet been associated with its cognate amino acid. M1 mutants are also deficient in the production of several additional transfer RNA species, as well as several larger RNAs. The significance of these results in relation to transfer RNA biosynthesis is discussed.     

Reduction of turgor induces rapid changes in leaf translatable RNA

The turgor of pea (Pisum sativum) leaves was reduced by exposing excised pea shoots to a stream of 23°C air for 20 min. Poly(A)⁺ RNA was isolated from control and wilted shoots, translated in vitro and radiolabeled translation products separated by electrophoresis on two-dimensional (isoelectric focusing-sodium dodecyl sulfate) polyacrylamide gels. This analysis showed that the levels of several poly(A)⁺ RNAs increased in wilted plants. Most of the poly(A)⁺ RNAs induced in wilted plants did not accumulate in response to heat shock or exogenously applied ABA even though endogenous ABA levels were found to increase in shoots 30 min after wilting and by 4 h had increased 50-fold (1 versus 0.02 microgram per gram fresh weight). A λgt10 cDNA library was constructed using poly(A)⁺ RNA from wilted shoots which had been incubated for 4 hours. Differential screening of the library identified four clones corresponding to poly(A)⁺ RNAs which are induced in wilted shoots.     

Nucleotide alterations in the bacteriophage T4 glutamine transfer RNA that affect ochre suppressor activity

We have determined the nucleotide sequences of the glutamine transfer RNAs that are coded by wild-type and psu₂⁺ ochre-suppressor strains of bacteriophage T4. The two transfer RNAs have the same sequence except for their anticodons, where NUG in the wild-type species is mutated to NUA in the psu₂⁺ species (N is a modified residue of U). This mutation is believed to confer suppressor activity on the psu₂⁺ glutamine tRNA. Three mutants derived from psu₂⁺ by loss of suppressor activity have been characterized with respect to their sequence alterations. Each mutant specifies a transfer RNA differing from the psu₂⁺ species by a nucleotide substitution that occupies a base-paired region in the cloverleaf arrangement of the molecule. The mutants synthesize a reduced amount of tRNA that is defective in nucleotide modifications and processing at the 5′ and 3′ termini.     

Enterococcus faecalis Gene Transfer under Natural Conditions in Municipal Sewage Water Treatment Plants

The ability of Enterococcus faecalis to transfer various genetic elements under natural conditions was tested in two municipal sewage water treatment plants. Experiments in activated sludge basins of the plants were performed in a microcosm which allowed us to work under sterile conditions; experiments in anoxic sludge digestors were performed in dialysis bags. We used the following naturally occurring genetic elements: pAD1 and pIP1017 (two so-called sex pheromone plasmids with restricted host ranges, which are transferred at high rates under laboratory conditions); pIP501 (a resistance plasmid possessing a broad host range for gram-positive bacteria, which is transferred at low rates under laboratory conditions); and Tn916 (a conjugative transposon which is transferred under laboratory conditions at low rates to gram-positive bacteria and at very low rates to gram-negative bacteria). The transfer rate between different strains of E. faecalis under natural conditions was, compared to that under laboratory conditions, at least 10⁵-fold lower for the sex pheromone plasmids, at least 100-fold lower for pIP501, and at least 10-fold lower for Tn916. In no case was transfer from E. faecalis to another bacterial species detected. By determining the dependence of transfer rates for pIP1017 on bacterial concentration and extrapolating to actual concentrations in the sewage water treatment plant, we calculated that the maximum number of transfer events for the sex pheromone plasmids between different strains of E. faecalis in the municipal sewage water treatment plant of the city of Regensburg ranged from 10⁵ to 10⁸ events per 4 h, indicating that gene transfer should take place under natural conditions.     

Complete mtDNA of Meretrix lusoria (Bivalvia: Veneridae) reveals the presence of an atp8 gene, length variation and heteroplasmy in the control region

The complete nucleotide sequence of the mitochondrial genome of the clam Meretrix lusoria (Bivalvia: Veneridae) was determined. It comprises 20,268 base pairs (bp) and contains 13 protein-coding genes, including ATPase subunit 8 (atp8), two ribosomal RNAs, 22 transfer RNAs, and a non-coding control region. The atp8 encodes a protein of 39 amino acids. All genes are encoded on the same strand. A putative control region (CR or D-loop) was identified in the major non-coding region (NCR) between the tRNA^Gly and tRNA^Gln. A 1087 bp tandem repeat fragment was identified that comprises nearly 11 copies of a 101 bp motif and accounts for approximately 41% of the NCR. The 101 bp tandem repeat motif of the NCR can be folded into a stem–loop secondary structure. Samples of eight individuals from Hainan and Fujian provinces were collected and their NCR regions were successfully amplified and sequenced. The data revealed a highly polymorphic VNTR (variable number of tandem repeats) associated with high levels of heteroplasmy in the D-loop region. The size of the CR ranged from 1942 to 3354 bp depending upon the copy number of the repeat sequence.     

A gradient of poly(A)+ RNA sequences in Xenopus laevis eggs and embryos

Xenopus laevis eggs and gastrula stage embryos were fractionated into three equal sections normal to the animal-vegetal axis, and poly(A)⁺ RNA was isolated from each section. Hybridization of these poly(A)⁺ RNAs with [³²P]cDNA synthesized using animal or vegetal poly(A)⁺ RNAs showed no detectable differences in the extents or rates of reaction. Thus, the vast majority of poly(A)⁺ RNAs are not segregated along the animal-vegetal axis. To increase the sensitivity of these experiments, [³²P]cDNAs were prepared which had reduced levels of RNA sequences from the animal region of the gastrula stage embryo or spawned unfertilized egg. Hybridization reactions with these probes showed that 3 to 5% of the input cDNA represents poly(A)⁺ RNA sequences enriched 2- to 20-fold in the vegetal region of the egg or gastrula stage embryo.     

A new pyrimidine-specific ribonuclease from bovine seminal plasma that is active on both single and double-stranded polyribonucleotides and that can distinguish between Mg2+-containing and Mg2+-depleted naturally occurring RNAs

The purification to homogeneity of a new ribonuclease, named RNAase SPL, from bovine seminal plasma is described. This nuclease, like the bovine pancreatic RNAase A, is pyrimidine specific. Its activity on single-stranded synthetic polyribonucleotides such as poly(rU) is significantly higher than that of RNAase A. However, unlike RNAase A, RNAase SPL is highly active on a double-stranded RNA such as poly[r(A · U)], and shows extremely limited activity on naturally occurring RNAs, such as Escherichia coli RNA, prepared with Mg²⁺ present throughout the isolation procedure. Under conditions of limiting hydrolysis in which RNAase A degrades 60 to 90% of total E. coli RNA to acid-soluble material and the remaining to material having a molecular weight lower than that of transfer RNA, RNAase SPL does not yield any acid-soluble products: it does not appear to degrade tRNA or 5 S RNA, and causes only a small number of nicks in the remaining RNAs to yield a limiting digest containing products with molecular weights ranging between 10,000 and 150,000. Absence of Mg²⁺ during the isolation procedure, or heat denaturation of the RNA makes it as susceptible to RNAase SPL as it is to RNAase A.The above and other related observations reported here support the view that there are Mg²⁺-dependent structural features, besides single and doublestrandedness, in naturally occurring RNAs, that can be distinguished by using the two nucleases RNAase SPL and RNAase A.     

Transfer RNA genes in the mitochondrial DNA of cytoplasmic petite mutants of Saccharomyces cerevisiae

Hybridization saturation analyses of mitochondrial DNA from 11 petite clones genetically characterized with respect to chloramphenicol and erythromycin resistance markers, have been carried out with 11 individual mitochrondrial transfer RNAs. Mitochondrial tRNA cistrons were lost, retained, or amplified in different petite strains. In some cases hybridization levels corrected for kinetic complexity of the mtDNA³ were two- to threefold greater than that for grande mtDNA indicating selective amplification, or increased number of copies, of the segment of mtDNA containing that tRNA cistron. Hybridization levels corrected for reduced kinetic complexity of petite mtDNAs in many cases were only 1 to 10% of that for grande mtDNA suggesting a low level of intracellular molecular heterogeneity of mtDNA with respect to tRNA cistrons. Some petite clones that retained tRNA genes continued to transcribe mitochondrial tRNAs, since tRNA isolated from these strains could be aminoacylated with Escherichia, coli synthetases and hybridized with mtDNA. Hybridization data allow us to order several of the tRNA cistrons on the mitochondrial genome with respect to the chloramphenicol and erythromycin antibiotic resistance markers.     

Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides

The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB) analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m¹A), N6-methyladenosine (m⁶A), pseudouridine, and 5-methylcytidine (m⁵C) showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m⁵C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m¹A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m⁶A antibody confirmed the demethylation of m⁶A by the specific demethylases fat mass and obesity-associated protein (FTO) and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m⁶A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism.     

Passive transfer of experimental allergic encephalomyelitis in the Lewis rat with activated spleen cells: differential activation with mitogens

It has previously been shown that spleen cell transfer of clinical EAE requires donor cells to be cultured in vitro prior to transfer. Donor cells must be stimulated when cultured, and either Con A or the encephalitogen, guinea pig myelin basic protein (BP), satisfies this stimulation requirement. Following recovery from passive disease, recipients of these in vitro cultured cells will subsequently develop clinical symptoms of EAE sooner than controls when challenged with BP in complete Freund's adjuvant (BP-CFA). In the present study, three T-cell mitogens were evaluated as donor cell stimulants in the required in vitro culture period. Pokeweed mitogen (PWM) as well as Con A stimulated the donor cell population to the degree that clinical EAE could be transferred with 5 × 10⁶ cultured viable cells. Con A at culture levels below 0.25 μg/ml did not yield transfer active cells even though proliferation levels were similar to those found at concentrations of Con A that did yield transfer active cells. Phytohemagglutinin (PHA)-stimulated cultures did not transfer clinical disease even though the degree of lectin induced proliferation ([³H]thymidine uptake as well as recovered cells from culture) was equivalent to the PWM- or Con A-stimulated, transfer positive, cultures. Mixing experiments suggested that the inability of PHA or low doses of Con A to induce transfer active cells was not due to the induction of suppressor cells. Although cells cultured with PHA do not transfer clinical EAE, recipients of these cells as well as recipients of either PWM- or Con A-stimulated donor cells develop an early appearance of disease upon subsequent challenge with BP-CFA. This included cells incubated with a concentration of Con A (0.1 μg/ml) which did not induce cells capable of transferring clinical EAE. These results suggest that PHA and perhaps the low dose of Con A may stimulate the proliferation of the EAE effector cell precursor population without causing the additional differentiation of this precursor population into the effector cell population which is capable of transferring clinical disease. Alternatively, PHA may expand only the helper cell population while effective doses of Con A and PWM would expand both helper and effector cell populations.     

Introduction and Expression of Cowpea Trypsin Inhibitor (CpTI) Gene in Transgenic Tobacco

A trypsin/chymotrypsin inhibitor (CpTI) was purified from mature cowpea seeds. A full length cDNA clone encoding the trypsin/chymotrypsin inhibitor protein was isolated from a cDNA library that was constructed using poly(A⁺) RNA from developing seeds. Tobacco protoplasts were transformed with a construct composed of CaMV 35S promoter, NPTII gene, CpTI gene, and NOS terminator, using polyethylene glycol (PEG)-mediated direct gene transfer. The CpTI gene has been integrated into the plant genome, and the expressed CpTI protein from transgenic plants was catalytically active.     

Mature methyl-deficient tRNA isolated from a mammary adenocarcinoma

A transplantable rat tumor, mammary adenocarcinoma 13762, accumulates tRNA which can be methylated in vitro by mammalian tRNA (adenine-1) methyltransferase. This unusual ability of the tumor RNA to serve as substrate for a homologous tRNA methylating enzyme is correlated with unusually low levels of the A₅₈-specific adenine-1 methyltransferase. The nature of the methyl-accepting RNA has been examined by separating tumor tRNA on two-dimensional polyacrylamide gels. Comparisons of ethidium bromide-stained gels of tumor vs. liver tRNA show no significant quantitative differences and no accumulation of novel tRNAs or precursor tRNAs in adenocarcinoma RNA. Two-dimensional separations of tumor RNA after in vitro [¹⁴C]methylation using purified adenine-1 methyltransferase indicate that about 25% of the tRNA species are strongly methyl-accepting RNAs. Identification of six of the tRNAs separated on two-dimensional gels has been carried out by hybridization of cloned tRNA genes to Northern blots. Three of these, tRNA^Lys3, tRNA^Gln and tRNA^Met_i, are among the adenocarcinoma methyl-accepting RNAs. The other three RNAs, all of which are leucine-specific tRNAs, show no methyl-accepting properties. Our results suggest that low levels of a tRNA methyltransferase in the adenocarcinoma cause selected species of tRNA to escape the normal A₅₈ methylation, resulting in the appearance of several mature tRNAs which are deficient in 1-methyladenine. The methyl-accepting tRNAs from the tumor appear as ethidium bromide-stained spots of similar intensity to those seen for RNA from rat liver; therefore, methyladenine deficiency does not seem to impair processing of these tRNAs.     

Nitrogen form, availability, and mycorrhizal colonization affect biomass and nitrogen isotope patterns in Pinus sylvestris

Nitrogen (N) isotope patterns are useful for understanding carbon and nitrogen dynamics in mycorrhizal systems but questions remain about how different N forms, fungal symbionts, and N availabilities influence δ¹⁵N signatures. Here, we studied how biomass allocation and δ¹⁵N patterns in Pinus sylvestris L. cultures were affected by nitrogen supply rate (3% per day or 4% per day relative to the nitrogen already present), nitrogen form (ammonium versus nitrate), and mycorrhizal colonization by fungi with a greater (Laccaria laccata) or lesser (Suillus bovinus) ability to assimilate nitrate. Mycorrhizal (fungal) biomass was greater with ammonium than with nitrate nutrition for Suillus cultures but similar for Laccaria cultures. Total biomass was less with nitrate nutrition than with ammonium nutrition for nonmycorrhizal cultures and was less in mycorrhizal cultures than in nonmycorrhizal cultures. The sequestration of available N by mycorrhizal fungi limited plant N supply. This limitation and the higher energetic cost of nitrate reduction than ammonium assimilation appeared to control plant biomass accumulation. Colonization decreased foliar δ¹⁵N by 0.5 to 2.2‰ (nitrate) or 1.7 to 3.5‰ (ammonium) and increased root tip δ¹⁵N by 0 to 1‰ (nitrate) or 0.6 to 2.3‰ (ammonium). Root tip δ¹⁵N and fungal biomass on root tips were positively correlated in ammonium treatments (r ²?=?0.52) but not in nitrate treatments (r ²?=?0.00). Fungal biomass on root tips was enriched in ¹⁵N an estimated 6–8‰ relative to plant biomass in ammonium treatments. At high nitrate availability, Suillus colonization did not reduce plant δ¹⁵N. We conclude that: (1) transfer of ¹⁵N-depleted N from mycorrhizal fungi to plants produces low plant δ¹⁵N signatures and high root tip and fungal δ¹⁵N signatures; (2) limited nitrate reduction in fungi restricted transfer of ¹⁵N-depleted N to plants when nitrate is supplied and may account for many field observations of high plant δ¹⁵N under such conditions; (3) plants could transfer assimilated nitrogen to fungi at high nitrate supply but such transfer was without ¹⁵N fractionation. These factors probably control plant δ¹⁵N patterns across N availability gradients and were here incorporated into analytical equations for interpreting nitrogen isotope patterns in mycorrhizal fungi and plants.