Poly(A)+ RNA and cytoskeleton during cyst formation in the cap ray of Acetabularia peniculus

Summary. The configuration and distribution of polyadenylated RNA (poly(A)⁺ RNA) during cyst formation in the cap rays of Acetabularia peniculus were demonstrated by fluorescence in situ hybridization using oligo(dT) as a probe, and the spatial and functional relationships between poly(A)⁺ RNA and microtubules or actin filaments were examined by immunofluorescence microscopy and cytoskeletal inhibitor treatment. Poly(A)⁺ RNA striations were present in the cytoplasm of early cap rays and associated with longitudinal actin bundles. Cytochalasin D destroyed the actin filaments and caused a dispersal of the striations. Poly(A)⁺ RNA striations occurred in the cytoplasm of the cap rays up to the stage when secondary nuclei migrated into the cap rays, but they disappeared after the secondary nuclei were settled in their positions. At that time, a mass of poly(A)⁺ RNA was present around each of the secondary nuclei and accumulated rRNA. This mass colocalized with microtubules radiating from the surface of each secondary nucleus and disappeared when the microtubules were depolymerized by butamifos, which did not affect the configuration of actin filaments. These masses of poly(A)⁺ RNA continued to exist even after the cap ray cytoplasm divided into cyst domains. Thus two distinct forms of poly(A)⁺ RNA population, striations and masses, appear in turn at consecutive stages of cyst formation and are associated with distinct cytoskeletal elements, actin filaments and microtubules, respectively. Correspondence and reprints: Graduate School of Kuroshio Science, Kochi University, 2-5-1 Akebono-cho, Kochi 780-8520, Japan.     

Nonradioactive in situ localization of poly(A)+ RNA during pollen development in anthers of tobacco (Nicotiana tabacum L.)

Summary A method is described for non-radioactive labeling of total mRNA [poly(A)+ RNA] in plastic-embedded plant tissue sections. Oligo-deoxythymidylic acid (oligo-dT) labeled with digoxigenin-conjugated dUTP was used for in situ hybridization to poly(A)+ RNA in sections of tobacco (Nicotiana tabacum) anthers. The digoxigenin was immuno-stained using antidigoxigenin IgG and gold-labeled protein-A, followed by silver enhancement of the gold label. Reproducibly similar positive staining patterns were obtained with digoxigenin-labeled oligo-dT and polyuridylic acid [poly(U)], but not with a similarly labeled sense probe, poly(A). In the developing anthers, from the onset of meiosis to the production of pollen grains, labeling patterns were compatible with a gradual depletion of nuclear and chromosome-associated sporophytic mRNA molecules during prophase of meiosis, followed by postmeiotic production of gametophytic mRNA in microspore nuclei and the vegetative nuclei of the pollen grains.Abbreviations BSA bovine serum albumin - DIG digoxigenin - IgG immunoglobulin-G - oligo-dT oligo-deoxythymidylic acid - PAS-ABB periodic acid Schiff-aniline blue black - PBS phosphate buffered saline - poly(A) polyadenylic acid - poly(U) polyuridylic acid - SSC standard saline citrate     

Synthesis and accumulation of ribosomes in individual cells of the female gametophyte of maize during its development

Summary The pattern of synthesis of ribosomes during three stages of development of the female gametophyte of maize has been studied by in situ hybridization using a ribosomal RNA probe. Changes in volume of individual cells of the embryo sac during its maturation have been determined by confocal microscopy. These data have permitted us to calculate the relative numbers of ribosomes in the cells of the embryo sac at different stages of their maturation. The egg apparatus and the central cell at all stages of development contain several fold greater numbers of ribosomes than are present in the antipodal cells or cells of the surrounding nucellus. The accumulation of ribosomes during embryo sac maturation appears to proceed at a constant and high rate, with the rate being highest in the developing central cell.     

A comparison of sequence complexity of nuclear and polysomal poly(A)+ RNA from different developmental stages ofXenopus laevis

Summary Nuclear poly(A)⁺ and polysomal poly(A)⁺ RNA were isolated from gastrula and early tadpole stages of the amphibianXenopus laevis. Complementary DNA was synthesized from all RNA preparations. Hybridization reactions revealed that at least all abundant and probably most of the less frequent nuclear and polysomal poly(A)⁺ RNA species present at the gastrula stage are also present at the early tadpole stage. On the other hand, there are nuclear RNA sequences at the latter stage which appear, if at all, only at lower concentrations at the gastrula stage. The polysomal poly(A)⁺ RNA hybridization reactions suggest the existence of polysomal poly(A)⁺ RNA sequences at early tadpole stages which are not present in the corresponding gastrula stage RNA.By cDNA hybridization with poly(A)^– RNA it could be shown that most of the poly(A)⁺ containing RNA sequences transcribed into cDNA were also present within the poly(A)^– RNA. It was estimated, that these sequences are 10 fold more abundant within the poly(A)^– polysomal RNA and 3–6 more abundant within the poly(A)^– nuclear RNA as compared to the poly(A)⁺ RNAs.     

Poly(a)+ rna during vegetative development ofacetabularia peniculus

Summary In the juvenile stage, the diploid giant-celled green algae Acetabularia spp. are differentiated into an upright stalk and an irregularly branched rhizoid. Early amputation and grafting experiments as well as biochemical and molecular analyses have shown that mRNA (as poly(A)⁺ RNA) is continuously supplied from the primary nucleus in the rhizoid and accumulates in the stalk apex. In the present study, localization of poly(A)⁺ RNA in the juvenile stage of theAcetabularia peniculus was investigated by fluorescent in situ hybridization using oligo(dT) as a probe. The signal was localized in the apical cytoplasm and, in addition, multiple longitudinal striations throughout the stalk and rhizoid cytoplasm. A large portion of the poly(A)⁺ RNA striations exhibited structural polarity, broadened at one end and gradually thinned toward the other end. Some of the striations in the rhizoid cytoplasm were continuous with a zone of signal in the area of the perinuclear rim. The poly(A)⁺ RNA striations were associated with thick bands of longitudinal actin bundles which run through the entire length of the stalk. Cytochalasin D caused fragmentation of the actin bundles and irregular distribution of the fluorescent signal. We suggest that the poly(A)⁺ RNA striations constitute a hitherto unknown form of packaged mRNA that is transported over large distances along the actin cytoskeleton to be stored and expressed in the growing apex.     

The size of poly(A)-containing RNAs in Drosophila melanogaster embryos

The size range of poly(A)-containing RNA from Drosophila melanogaster embryos has been estimated by hybridization with ³H-labeled poly(U) and subsequent fractionation on sucrose gradients. The median size of nuclear poly(A)-containing RNA is about 30 S (6000 nucleotides), and the median size of cytoplasmic poly(A)-containing RNA is about 17 S (1800 nucleotides). The relationship of these sizes to messenger RNA needed to code for protein and to the length of DNA contained in a chromomere is discussed.Research grant support was provided by NIH (6M35558; HD-00266) and NSF (GB-30600).     

Spatial distribution of mRNA during pre-fertilization ovule development in Capsella bursa-pastoris

Summary In situ hybridization of sections of the ovary and ovule of Capsella bursa-pastoris with ³H-polyuridylic acid [³H-poly(U)] showed the presence of polyadenylic acid-containing RNA [poly(A) + RNA] in the cells of the placenta and nucellus. During megasporogenesis there was a decrease in ³H-poly(U) binding activity of the nucellar cells concomitant with the appearance of poly(A) + RNA in the integuments. As the typical eight-nucleate embryo sac was formed, ³H-poly(U) binding was not apparent around the quartet of nuclei at the chalazal end, while it persisted at the micropylar end. Both the egg and synergids as well as the chalazal proliferating tissue showed high concentrations of poly(A) + RNA in their cytoplasm. The results suggest a role for transient localizations of poly(A) + RNA during female sporogenesis and gametogenesis in C. bursa-pastoris.     

Regulation of poly(A) polymerase activity and poly(A)+ RNA in germinated wheat embryos under conditions of pathogenesis

The induction of poly(A) polymerase was accompanied by a rise in the level of poly(A)⁺ RNA during early germination of excised wheat embryos (48 h). Fractionation of this RNA-processing enzyme by acrylamide gel electrophoresis and also by molecular sieving on Sephadex G-200 revealed a single molecular form of poly(A) polymerase with a molecular weight of 125 000. Wheat poly(A) polymerase specifically catalyzed the incorporation of [³H]AMP from [³H]ATP into the polyadenylate product only in the presence of primer RNA. Substitution of [³H]ATP by other labelled nucleoside triphosphates, such as [³H]GTP, [³H]UTP or [α-³²P]CTP in the assay mixture did not yield any labelled polynucleotide reaction product. The ³H-labelled reaction product was retained on poly(U)-cellulose affinity column and was not degraded by RNAase A and RNAase T₁ treatment. In addition, the nearest-neighbour frequency analysis of the ³²P-labelled reaction product predominantly yielded [³²P]AMP. Thus, characterization of the reaction product clearly indicated its polyadenylate nature. The average chain length of the [³H]poly(A) product was 26 nucleotides. Infection of germinating wheat embryos by a fungal pathogen (Drechslera sorokiana) brought about a severe inhibition (62–79%) of poly(A) polymerase activity. Concurrently, there was a parallel decrease (73%) in the level of poly(A)⁺ RNA. Inhibition of poly(A) polymerase activity in infected embryos could be due to enzyme inactivation, which in turn brought about a downward shift in the level of poly(A)⁺ RNA. The crude extract of the cultured pathogen contains a non-dialysable, heat-labile factor, which, along with a ligand, inactivates (65–74%) poly(A) polymerase in vitro. The fungal extracts also contained a dialysable, heat-stable stimulatory effector which activated wheat poly(A) polymerase (3.6–4.0-fold stimulation) in vitro. However, the stimulatory fungal effector was not expressed in vivo, but was detectable after the inhibitory fungal factor had been destroyed by heat-treatment in our in vitro experiments.     

Localization of Escherichia coli poly(A) polymerase I in cellular membrane

Poly(A) polymerase I (PAP I), the pcnB gene product, is the main enzyme responsible for RNA polyadenylation in Escherichia coli. Polyadenylated RNA molecules are rapidly degraded by a multiprotein complex called RNA degradosome. Here we demonstrate that apart from its presence in cytosol, PAP I is also localized in cellular membrane. Although this observation might appear surprising, it was demonstrated recently by others that E. coli RNA degradosome is also associated with the cytoplasmic membrane. Moreover, we show that development of single-stranded RNA bacteriophages MS2 and Qbeta, but not that of single-stranded DNA bacteriophage M13, is more efficient in the pcnB mutant relative to an otherwise isogenic pcnB(+) host.     

Cloning of cDNA sequences derived from poly(A)+ nuclear RNA ofXenopus laevis at different developmental stages: Evidence for stage specific regulation

Summary Nuclear poly(A)⁺ RNA was isolated from gastrula and early tadpole stages ofXenopus laevis, transcribed into cDNA and integrated as double stranded cDNA by the G-C joining method into the Pst cleavage site of plasmid pBR 322. After cloning inE. coli strain HB 101 the clone libraries were hybridized to³²P labelled cDNA derived from nuclear poly(A)⁺ RNA of the two different developmental stages. About 20% of the clones gave a positive hybridization signal thus representing RNA molecules of high and medium abundance. From these clones, some individual clones were identified containing sequences which are not present at the oocyte and gastrula stages but which are transcribed at the early tadpole stage of embryonic development.     

Intracellular levels of polyadenylated RNA and loss of vigour in germinating wheat embryos

Polyadenylated-RNA (Poly(A)⁺RNA) levels have been studied during the germination of wheat embryos of high viability but differing vigour. In high-vigour embryos imbibed at 20°C the level of poly(A)⁺RNA falls dramatically over the first hour of imbibition, then remains constant up to 3 h of imbibition before increasing rapidly to a level similar to that found in the quiescent state by 7 h of imbibition. Median-vigour embryos imbibed at 20°C show similar changes in poly(A)⁺RNA content but the initial decrease and subsequent increase in poly(A)⁺RNA levels are less marked. On imbibition at 10°C, the poly(A)⁺RNA content in high-vigour embryos decreases to a lesser extent during the first hour than at 20°C and the level increases more slowly over the next 6 h than during the same time period at 20°C. The level of poly(A)⁺RNA in medianvigour embryos remains constant over the first 4 h of germination and then falls to a level of about half that found in quiescent high-vigour embryos. Polyacrylamide gel electrophoresis of total-RNA samples shows that the polyadenylic acid (poly(A)) sequences occur in RNA species ranging in size from 35-7S. Polyacrylamide gel electrophoresis of isolated poly(A) sequences demonstrates the presence of two size classes of poly(A) in quiescent embryos, but at 20°C a more heterodisperse pattern appears by 2 h of imbibition. At 10°C, two size classes of poly(A) persist throughout the period studied in both high- and median-vigour embryos, although in median-vigour embryos the ratio of larger: smaller poly(A)-tail sizes decreases more rapidly than in high-vigour embryos.Abbreviations Poly(A) polyadenylic acid - poly(U) polyuridylic acid - poly(A)⁺RNA polyadenylated RNA     

Polyribosome formation and poly(A)-containing RNA in embryos of the sand dollar,Dendraster excentricus

Summary The pattern of appearance of ribosomes, newly synthesized mRNA, and poly(A)-containing mRNA in polyribosomes has been examined in sand dollar embryos. From early blastula until shortly before hatching small polyribosomes engaged in histone synthesis predominate. At the time of hatching, when the rate of cell increase is maximal, the proportion of poly(A)-containing RNA in polyribosomes is low. After hatching a new class of large polyribosomes appears and the amount of poly(A)-containing polyribosomal RNA increases. Cordycepin, an inhibitor of RNA adenylylation, prevents the appearance of the large polyribosomes after hatching as well as the increase in poly(A)-containing polyribosomal RNA.     

Altered development of polysomal RNA activity in chick-pea (Cicer arietinum L.) embryonic axes. Effects of abscisic acid and temperature

The in vitro activity of polysomal polyadenylated RNA (poly(A)RNA) was studied using chick-pea (Cicer arietinum L.) embryonic axes subjected to treatments retarding germination (H₂O 30°C and abscisic acid [ABA] 30°C) or inducing a false germination (thiourea 30°C) in which normal protein synthesis and growth did not occur. All treatments induced a smaller proportion of poly(A)RNA compared with the control (H₂O 25°C). However, poly(A)RNA obtained in the presence of ABA had a similar in vitro activity to that of the control. The translation of mRNA from embryonic axes germinated at high temperatures was extensively blocked (70%) by methyl-7-guanosine-5-triphosphate, whereas mRNA translation from axes treated with H₂O-25°C and ABA was completely blocked (100%), indicating a greater cap dependence in the latter cases. Polyacrylamide gel electrophoresis showed that ABA and H₂O-30°C each induced the synthesis of a polypeptide with an approximate M_r of 32 kDa, probably a germination regulator. It is suggested that ABA and high temperatures could regulate germination at the translational level as well as affecting ionic-exchange properties, as has been previously demonstrated (Hernández-Nistal et al. 1983, Physiol. Plant. 57, 273–278).Abbreviations ABA abscisic acid - Poly (A) RNA polyadenylated RNA - TU thiourea     

In situ hybridization of corticotropin-releasing factor-encoding messenger RNA in the hypothalamus of the white sucker,Catostomus commersoni

Summary In situ hybridization procedure with a ³²P-labelled synthetic oligonucleotide probe was used to detect corticotropin-releasing factor-encoding messenger RNA (CRF mRNA) in the hypothalamus of the white sucker, Catostomus commersoni. Adjacent sections were immunostained by a sucker CRF-specific antiserum. CRF mRNA-containing neurons were identified by autoradiography in the magnocellular and parvocellular subdivisions of the preoptic nucleus (PON). Many of these neurons were also immunostained by sucker antiserum, showing the same distribution patterns. These results confirm the presence of CRF mRNA and CRF peptide in the white sucker hypothalamus and support the view that the magnocellular and parvocellular neurons of the PON may be involved in the control of adrenocorticotropic hormone secretion from the pituitary in the white sucker.     

Structure and function of the microtubular cytoskeleton during megasporogenesis and embryo sac development in Gasteria verrucosa (Mill.) H. Duval

Summary Using immunocytochemical techniques, tubulin distribution in various stages of meiosis and embryo sac development was studied. In the archespore cell some microtubules appeared to be randomly oriented. During zygotene and pachytene, when the cell volume increases, a large number of microtubules in dispersed configurations and bundles were observed. During this stage the nucellar cells divide, and their parallel cortical microtubules play an important role in preparing the direction of cell enlargement. The protoderm cells show anticlinal-directed cortical microtubules. It can be concluded that the enlargement of the meiocyte during these early meiotic stages is influenced both by its own cytoskeleton and by growth of the nucellus. Thereafter, the microtubules function directly in meiosis and disappear for the greater part until the two-nucleate coenocyte is formed. In a four-nucleate coenocyte microtubules reappear around the nucleus; in a young synergid, randomly oriented microtubules are involved in cell shaping during the formation of the filiform apparatus; in the synergids of the mature embryo sac, many parallel arrays of microtubules are present. Microtubules are less abundant in other cells. It is concluded that the cytomorphogenesis of the developing coenocyte and embryo sac are due to cell growth of the nucellar cells together with vacuolation of the coenocyte.     

Genomic in situ hybridization inArachis (Fabaceae) identifies the diploid wild progenitors of cultivated (A. hypogaea) and related wild (A. monticola) peanut species

Genomic in situ hybridization offers a powerful tool for investigating genome organisation and evolution of taxa known, or suspected, to be allopolyploids. The question of the diploid progenitors of cultivated peanut (Arachis hypogaea, 2n=4x=40) has been the subject of numerous studies at cytogenetical, cytochemical, biochemical and molecular levels, but no definitive conclusions have been reached. The biotinylated total genomic DNA from potential diploidArachis species were separately hybridized in situ to root tip chromosomes ofA. hypogaea and wild speciesA. monticola (2n=4x=40) without or mixed with an excess of unlabelled DNA from the species not used as a probe. Among the range of different species combinations used, the strong and uniform signals given by labelledA. ipaensis DNA when hybridized toA. hypogaea andA. monticola in combination with unlabelledA. villosa DNA indicates that overall molecular composition of twenty chromosomes ofA. hypogaea andA. monticola is very similar toA. ipaensis chromosomes. ProbingA. hypogaea andA. monticola chromosomes with labelled genomic DNA fromA. villosa mixed with unlabelled DNA fromA. ipaensis likewise labelled strongly and uniformly the other twenty chromosomes. BarringA. ipaensis, all the diploidArachis species presently investigated had characteristic centromeric bands in the twenty chromosomes within the complement indicating a clear division ofA. ipaensis from other species. InA. hypogaea andA. monticola only twenty chromosomes showed centromeric bands. These results (i) confirm the allopolyploid nature ofA. hypogaea andA. monticola, (ii) strongly support the view that wildA. monticola and cultivatedA. hypogaea are very closely related, and (iii) indicate thatA. villosa andA. ipaensis are the diploid wild progenitors of the tetraploid species studied. The present results also reveal that the nucleolus organizing region (NOR) originating fromA. villosa alone is expressed in the two tetraploid species.