Polyadenylate-containing RNA in dormant and germinating Botryodiplodia theobromae pycnidiospores.

Hybridization of [3H]polyuridylic acid to RNA isolated from Botryodiplodia theobromae pycnidiospores yielded an estimate of about 6.25 x 10(5) polyadenylate-containing RNA (poly A(+) RNA) molecules per dormant spore. The number increased about fourfold by the time of germ tube emergence at 3 h. The average size of this presumed mRNA was about 4.1 x 10(5) daltons (1275 nucleotides), with an average polyadenylate segment length of 26 nucleotides. Neither of these values changed significantly during germination. The earliest detectable (first 30 min of germination) de novo synthesized mRNA's were rapidly incorporated into polyribosomes. This newly synthesized, presumably functional, mRNA was composed of both poly A(+) RNA and polyadenylate-lacking RNA. The average sizes of the two polyribosomal mRNA subpopulations and the total poly A(+) RNA population were identical.     

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.     

A temporal analysis of the synthesis of the mRNA sequestered in zoospores of Blastocladiella emersonii

To determine when the dormant mRNA of Blastocladiella emersonii zoospores is synthesized, the metabolism of poly(A) RNA and rRNA was studied during growth and sporulation using pulse-chase techniques. Zoospore poly(A) RNA is synthesized at all stages of the growth cycle investigated in cultures grown either on a normal 15-hr growth cycle or in minicyclic cultures induced to sporulate after only 6.5 hr growth. For cells labeled during the growth phase the specific activity of the pulse-labeled poly(A) RNA and rRNA was identical at the beginning and end of sporulation for any of the 2-hr labeling times investigated. From this it was concluded there is neither a preferential conservation nor degradation during sporulation of the poly(A) RNA and rRNA synthesized at various times during growth. Poly(A) RNA synthesized during early sporulation is preferentially degraded; in contrast, poly(A) RNA synthesized during late sporulation is conserved in the zoospore. Approximately one-third of the total zoospore poly(A) RNA accumulates during the final 15–20 min of sporulation. The accumulation rate for both poly(A) RNA and rRNA decreases as sporulation proceeds. In addition, the rate of degradation for both types of RNA decreases at later stages of sporulation.     

The polyadenylated RNA of zoospores and growth phase cells of the aquatic fungus,Blastocladiella

The stored poly(A) + RNA from zoospores of the aquatic fungus Blastocladiella emersonii represents 2.5% of the total RNA and has a model MW of 425,000 daltons and an average poly(A) isostich of 32 bases. The poly(A) + RNA also represents 2.5% of the total RNA from early growth phase cells and has a modal MW of 360,000 daltons and an average poly(A) isostich of 38 bases. The poly(A) + RNA from spores and 2-hr plants contains a structure resistant to RNases T₁, T₂, and A, which can be labeled with ³²PO₄ and which will bind to DBAE-cellulose. These characteristics strongly suggest that both the zoospore poly(A) + RNA and the 2-hr cell poly(A) + RNA are capped at the 5′ end; and, hence, it is unlikely that capping is involved in the control of protein synthesis during germination.Approximately 80% of the poly(A) + RNA of the spore is located in the membrane-enclosed ribosomal nuclear cap, and more than 90% of the poly(A) + RNA within the cap is found in the 80S monoribosome and heavier fractions.Synthesis of new poly(A) + RNA occurs very early during zoospore germination, and the labeled poly(A) + RNA rapidly enters the newly organized polysomes. The labeling data for early germination also suggest that cytoplasmic polyadenylation occurs.     

Polyadenylic acid on poliovirus RNA. III. In vitro addition of polyadenylic acid to poliovirus RNAs.

A crude RNA polymerase preparation was made from HeLa cells infected for 3 h with poliovirus. All virus-specific RNA species labeled in vitro (35S RNA, replicative intermediate RNA [RI], and double-stranded RNA [dsRNA]) would bind to poly(U) filters and contained RNase-resistant stretches of poly(A) which could be analyzed by electrophoresis in polyacrylamide gels. After incubation for 45 min with [3-H]ATP in the presence of the other three nucleoside triphosphates, the labeled poly(A) on the RI and dsRNA migrated on gels as relatively homogenous peaks approximately 200 nucleotides in length. In contrast, the poly(A) from the 35S RNA had a heterogeneous size distribution ranging from 50 to 250 nucleotides. In the absence of UTP, CTP, and GTP, the size of the newly labeled poly(A) on the dsRNA and RI RNA was the same as it was in the presence of all four nucleoside triphosphates. However the poly(A) on the 35S RNA lacked the larger sequences seen when the other three nucleoside triphosphates were present. When [3-H]ATP was used as the label in infected and uninfected extracts, heterogeneous single-stranded RNA sedimenting at less than 28S was also labeled. This heterogeneous RNA probably represents HeLa cytoplasmic RNA to which small lengths of poly(A) (approximately 15 nucleotides) had been added. These results indicate that in the in vitro system poly(A) can be added to both newly synthesized and preexisting RNA molecules. Furthermore, an enzyme capable of terminal addition of poly(A) exists in both infected and uninfected extracts.     

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     

Changes in mRNA of Vigna mungo Cotyledons during Seed Germination

Quantitative and qualitative changes of mRNA in Vigna mungocotyledons during seed germination have been investigated. TotalRNA is higher in dry cotyledons and declines during germination.Poly(A)⁺ RNA also is present at a relatively high level in drycotyledons, increases slightly during the first day of germination,and then decreases. Polysomal RNA is very low in dry cotyledonsbut increases rapidly during the first day of germination, andthen declines. The translational activity of the mRNA in a wheatgerm cell-free system is low on day 0 but increases rapidlyon day 1 of germination. Two-dimensional gel electrophoresisof in vitro translation products reveals that many new peptidesare synthesized on day 1 of germination. Synthesis of most ofthese polypeptides continue throughout 5 days of germination. Change in the mRNA population during germination has been investigatedusing cDNA against poly(A)⁺ RNA from 3-day-old cotyledons. Withtotal RNA of day 3 and 5, the cDNA strongly hybridized withRNA similar in size to 25 S ribosomal RNA, but no specific bandsare detected with samples of day 0 or 1. With poly(A)⁺ RNA ofday 5 or 1, the cDNA tends to hybridize with RNAs of relativelysmall molecular size. Cordycepin and -amanitin prevent the increasein poly (A)⁺ RNA content and the appearance of new mRNAs duringthe first day of germination. ¹Present address: Division of Regulation of Macromolecular Function,Institute for Protein Research, Suita City, Osaka 565, Japan. (Received January 13, 1986; Accepted June 10, 1986)     

Reactivation of Ribonucleic Acid and Protein Synthesis During Germination of Blastocladiella Zoospores and the Role of the Ribosomal Nuclear Cap

Freshly harvested zoospores of Blastocladiella emersonii begin to germinate about 15 min after inoculation into a defined growth medium at a density of 10(6) zoospores per ml. Flagellum retraction accompanies encystment, and dispersal of the ribosomal nuclear cap takes place shortly thereafter. The primary rhizoid begins to emerge at 25 to 30 min and starts to branch at ca. 60 min. The first nuclear division occurs between 120 and 190 min. The dry weight per cell increases linearly after 60 min, whereas the deoxyribonucleic acid per cell doubles between 120 and 240 min. A linear increase in total ribonucleic acid (RNA) is detectable beginning at 40 to 45 min, and in total protein beginning at 80 min; neither process is interrupted during nuclear division. Encystment and nuclear cap disorganization are associated with a sharp rise in the rates of precursor incorporation into RNA and protein. Cycloheximide at 20 mug/ml prevents leucine incorporation at all stages and inhibits development beyond the earliest encystment stage. Actinomycin D at 25 mug to 50 mug/ml prevents uracil incorporation, but it has no effect on leucine incorporation or development until 40 to 45 min. At the latter stage, actinomycin D causes a sharp developmental arrest and begins to inhibit leucine incorporation. It is concluded that early protein synthesis must occur on the ribosomes formed during the prior growth phase and conserved through the zoospore stage in the nuclear cap. The results further indicate that this synthesis is dependent upon messenger RNA already present in the zoospore before germination.     

Mobilization of newly synthesized RNAs into polysomes inXenopus laevis embryos

Summary The mobilization of newly synthesized 18S and 28S rRNAs, 4S RNA and poly(A)⁺ RNA into polysomes was studied in isolated cells ofXenopus laevis embryos between cleavage and neurula stages. Throughout these stages, 4S RNA and poly(A)⁺ RNA were mobilized immediately following their appearance in the cytoplasm. 18S rRNA however, stayed in the ribosomal subunit fraction for about 30 min until the 28S rRNA appeared, when the two rRNAs were mobilized together at an equimolar ratio. This mobilization, at a 1:1 molar ratio, appeared to be realized at initiation monome formation. Thus, the efficiency of the mobilization of two newly synthesized rRNAs, shortly after their arrival at the cytoplasm, differed considerably but difference disappeared once steady state was reached.The contribution of newly synthesized 18S and 28S rRNAs to polysomes remains small throughout early development. around 3% of newly synthesized 4S RNA is polysomal which is the same distribution observed for unlabeled 4S RNA. Less than 10% of the newly synthesized cytoplasmic poly(A)⁺ RNA was mobilized into polysomes during cleavage, but in later stages the proportion increased to around 20%–25%. These results show that newly synthesized RNAs are utilized for protein synthesis at characteristic rates soon after they are synthesized during early embryonic development. On the basis of the data presented here and elsewhere we discuss quantitative aspects of the utilization of newly synthesized and maternal RNAs during early embryogenesis.     

Polyadenylic acid-containing RNA and its polyadenylic acid sequences newly synthesized in isolated embryonic cells of Xenopus laevis.

Newly synthesized polyriboadenylic acid [poly(A)]-containing RNA and its poly(A) sequences were isolated and characterized in Xenopus embryonic cells. Upon sedimentation analysis, the poly(A)-containing RNA labeled for 30 min showed a very heterogeneous size distribution ranging from 9 to >40 S. After 5 hr of labeling, the profile became much less heterogeneous and the main component was distributed in the 9–28 S region. The average molecular weight of 6.5–7.0 × 10⁵ daltons was calculated for the 5-hr labeled RNA. This poly(A)-containing RNA, comprising about 10% of the total labeled RNA, was metabolically stable and accumulated linearly for 5 hr. Gel electrophoresis of the RNA revealed the presence of little or no free poly(A) sequences. Most of the poly(A) sequences, which were isolated from 30-min labeled poly(A)-containing RNA migrated as a single discrete component approximately 150 nucleotides long. In contrast, they were slightly smaller (130 nucleotides long) and more heterogeneous, when obtained from the poly(A)-containing RNA labeled for 5 hr. From these results, it may be likely that the embryonic poly(A)-containing RNA is similar in size to the steady-state population of the poly(A)-containing RNA reported to occur in vitellogenic oocytes and cultured kidney cells of the same species.     

Newly Synthesized mRNA Is Translated during the Initial Imbibition Phase of Germinating Maize Embryo

RNA synthesis is activated in the cells of the plant embryo very soon after the start of seed imbibition. We previously reported that mainly heterogeneous nuclear RNA is synthesized in the radicle of Zea mays embryo during the first hours of germination. The present study was undertaken in order to detect the time of appearance of the newly synthesized messenger RNA in the polysomes of germinating maize axes.

Free polysomes were prepared from embryonic axes rehydrated for 2 hours in the presence of radioactively labeled uridine. These polysomes were shown to be labeled and to contain labeled particles sedimenting, after dissociation with EDTA, in the 10S to 40S region of a sucrose gradient. The labeled polysomal RNA migrates heterogeneously in a gel with a mean size corresponding to about 16S, and 60% of these molecules are polyadenylated.

The data indicate that the newly synthesized RNA associated with the polysomes after 2 h of germination consists of messenger RNA molecules. Analysis of the polysomes prepared 0.5 and 1 h after the start of imbibition suggests that translation of the newly synthesized messenger RNA probably occurs within the 1st hour of imbibition of the isolated axis, thus well before the completion of the initial water uptake.

     

The occurrence and distribution of poly(a) ribonucleic Acid in soybean

The occurrence and distribution of poly(A) sequences in the RNA of soybean (Glycine max var. Wayne) have been studied. Only one of the two species of AMP-rich RNA contains poly(A). D-RNA does not contain detectable poly(A) sequences. The TB-RNA is the poly(A) RNA in this system. At least a part (up to 50% or more) of the mRNA in polyribosomes contains a poly(A) sequence. The poly(A) RNA is heterodisperse in size but has a mean size of approximately 18S (2,000 nucleotides) in urea and formamide gels. The poly(A) fragment resulting from ribonuclease A and T₁ digestion migrates as a broad band overlapping the 4 to 5.8S regions of the gels with a mean size of somewhat greater than 5S. No evidence was found for the occurrence of a discrete oligo(A) fragment in the poly(A) RNA; however, oligonucleotides which migrate faster than the poly(A) fraction were observed in preparations which were not bound to oligo(dT) cellulose prior to electrophoresis. This oligonucleotide region was enriched in AMP (up to about 65%) as would be expected after ribonuclease A and T₁ digestion.     

Poly(A)+ RNA synthesis in germinating pollen ofNicotiana tabacum L

Poly(A)⁺RNA is synthesized during the first hours of pollen germination and is rapidly incorporated into polysomal structures. After a 2-h pulse with uracil-¹⁴C, 42% of the transcribed fraction of polysomal RNA is polyadenylated. Following 4 h of germination the amount of the newly-made poly(A)⁺RNA decreases steadily at the rate of about 14% per h, whereas that of rapidly-labelled poly(A)⁻RNA continues to grow. Beginning 1 h of cultivation the ratio of poly(A)⁻/poly(A)⁺RNA increases exponentially. Similarly as in non-polyadenylated mRNA the main portion of the synthesized polysomal poly(A)⁺RNA sediments at a rate of 4 to 14 S and its mean size decreases slightly with the time of labelling. RNA isolated from nuclei and cell wall containing pollen tube fraction differed from the polysomal one in higher apeoific radioactivity and the polyadenylated RNA exhibited higher size distribution. The comparison of the results with earlier observations suggests the involvement of poly(A)in mRNA translation in pollen tubes.