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.     

Use of a cloned library for the study of abundant poly(A)+RNA during Xenopus laevis development

Over 200 cloned sequences from recombinant DNA libraries prepared from Xenopus laevis embryonic poly(A)⁺RNA have been analyzed by colony hybridization with [³²P]cDNA prepared from poly(A)⁺RNA from several stages of development. The period of early embryogenesis extending through the beginning of gastrulation (stage 10) is marked by the relative constancy of the abundant poly(A)⁺RNA population. Between the gastrula and tailbud stages (stage 24) there is a dramatic change in the pattern of abundant poly(A)⁺RNA species; the new pattern remains fairly constant for at least 2 days of development to the late prefeeding tadpole stages (stage 41). We have also compared nonpolysomal and polysomal poly(A)⁺RNA populations at two different stages. In stage 10 (early gastrula) postribosomal (free ribonucleoprotein) and polysomal poly(A)⁺RNA populations partly overlap; however, many cloned sequences occur in quite different concentrations in one fraction or the other. Among the sequences that are predominantly nonpolysomal at gastrula few become predominantly polysomal at tailbud stages. Thus, we have no evidence for a major recruitment of abundant nonpolysomal RNAs into polysomes with progressing development. We rather observe a general pattern in which a cloned sequence that is nonpolysomal in one stage of development tends to be nonpolysomal (if detectable at all) in other stages as well.     

Mitochondrial RNAs are abundant in the poly(A)+RNA population of early frog embryos

Mitochondrial sequences have been identified within a set of cloned complementary DNAs that had been copied from poly(A)⁺RNA of two embryonic stages of Xenopus laevis (Dworkin and Dawid, 1980, Dworkin and Dawid, 1980, Develop. Biol.76, 435–448 and 449–464). Mitochondrial sequences were found to be highly abundant in gastrula stage poly(A)⁺RNA sequences; in tadpole RNA their relative abundance is reduced severalfold. Mitochondrial sequences account for the most abundant poly(A)⁺RNA molecules in the gastrula population. The high abundance of mitochondrial RNA in early stages may be the consequence of the accumulation of large numbers of mitochondria in the egg.     

Electrophoretic analysis of newly synthesized and stored maternal RNA during oogenesis ofCalliphora erythrocephala (Dipt.)

Summary Ovaries ofC. erythrocephala synthesize large amounts of poly(A)⁺ and poly(A)^– RNA during early and middle stages of oogenesis as shown by labelling with³H-uridine in vivo. After incubation for 1 h, a striking difference in the electrophoretic pattern of newly synthesized labelled poly(A)⁺ RNA and the poly(A)⁺ RNA present in sufficient amounts for optical density measurements (steady state poly(A)⁺ RNA) was observed. During early and mid-oogenesis, in the poly(A)^– RNA fraction, 4S predominantly mature rRNA, 5S RNA and tRNA were labelled. These fractions were no longer synthesized during late oogenesis, whereas poly(A)⁺ RNA was labelled continously During oogenesis stage specific differences in the size distribution of newly synthesized and steady state poly(A)⁺ RNA were not obvious. However, different sizes of labelled poly(A)⁺ RNA species were detected in 0–2h old preblastoderm embryos, after injection of³H-uridine into females either 3–4 days (stage 3–4 of oogenesis) or 24 h before oviposition (stage 5–6 of oogenesis). This difference in RNA synthesis was related to the presence of active nurse cell nuclei. The poly(A)⁺ RNA fraction represents about 2–3% of the total RNA in both ovaries and freshly laid eggs as judged by measurements of optical density and radioactivity bound to oligo(dT). The length of poly(A)-segments in ovarian poly(A)⁺ RNA varied from about 30 to 200 nucleotides.     

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.     

Poly(A+)mRNA sequences in free mRNP and polysomes of mouse ascites cells

The poly(A⁺)RNA of the free mRNP of mouse Taper ascites cell contains a very reduced number of different mRNA sequences compared to the polysome poly(A⁺)RNA. By the technique of mRNA:cDNA hybridization we have determined that the free mRNP contains approximately 400 different mRNA sequences while the polysomes contain about 9000 different mRNAs. The free mRNP poly(A⁺)RNA sequences are present in two abundance classes, the abundant free mRNP class containing 15 different mRNA sequences and the less abundant free mRNP class containing 400 different mRNAs. The polysome poly(A⁺)RNA consists of three abundance classes of 25, 500 and 8500 different mRNA sequences.Despite its intracellular location in RNP structures not directly involved in protein synthesis the poly(A⁺)RNA purified from the free RNP of these cells was a very effective template for protein synthesis in cell-free systems. Cell-free translation products of free mRNP and polysome poly(A⁺)RNAs were analyzed by two-dimensional gel electrophoresis. This analysis confirmed the hybridization result that the free mRNP poly(A⁺)RNA contained fewer sequences than polysomal poly(A⁺)RNA. The abundant free RNP-mRNA directed protein products were a subset of the polysome mRNA-directed protein products. The numbers of more abundant products of cell-free protein synthesis directed by the free RNP-mRNA and polysomal mRNA were in general agreement with the hybridization estimates of the number of sequences in the abundant classes of these two mRNA populations.     

Comparison of polysomal polyadenylated RNA from embryonal carcinoma and committed myogenic and erythropoietic cell lines

Using the technique of mRNA-cDNA hybridization, we have examined the polysomal poly(A)⁺ mRNA base-sequence complexity in three different mouse cell lines: mouse embryonal carcinoma cells, myoblast cells and Friend erythroleukemic cells. These cells express 7700, 13,200 and 6200 mRNA sequences, respectively, distributed in three frequency classes. Reciprocal heterologous hybridization experiments revealed that there is a large degree of homology, a subset of 6000 common sequences being present on the polysomes of all three cell types. Myoblast mRNA is capable of hybridizing all reactive embryonal carcinoma cell cDNA, with kinetics close to the homologous embryonal carcinoma cell curve, thus indicating that all embryonal carcinoma cell sequences are present on myoblast polysomes, the majority at similar abundance. Conversely, embryonal carcinoma cell mRNA fails to hybridize 12% of myoblast cDNA, apparently arising primarily from the complex frequency class. This was confirmed by using myoblast fractions partially enriched in abundant and rare sequences. As a proportion of the rare class, this 12% fraction represents about 4500 sequences close to the difference in base-sequence complexity between myoblast and embryonal carcinoma cells.Homologous and heterologous hybridization with total and fractionated Friend cell cDNA probes revealed that all Friend cell polysomal poly(A)⁺ RNA sequences are common to embryonal carcinoma cell polysomes—apart from a small group of sequences drawn from the abundant class, corresponding to about 10% of Friend cell cDNA. This represents about 12 sequences from the abundant class. In addition, certain common sequences in the abundant Friend cell frequency class are present at lower frequency in embryonal carcinoma cell polysomes. Friend cell polysomal poly(A)⁺ RNA fails to hybridize 7–10% embryonal carcinoma cell cDNA apparently derived from the rare frequency class. As a fraction of the rare class, this corresponds approximately to the difference (about 1500 sequences) in complexity between the Friend and embryonal carcinoma cell lines.     

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.     

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.     

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.     

Evolutionary conservation of DNA sequences expressed in sea urchin eggs and early embryos

Summary DNA sequence divergence measurements indicate thatStrongylocentrotus franciscanus is more distinct fromS. purpuratus andS. drobachiensis than these two species are from each other, in agreement with paleontological and morphological evidence. The evolutionary divergence of several classes of expressed DNA sequences was compared with that of total single-copy DNA. BetweenS. franciscanus andS. purpuratus the divergence of cDNA made from gastrula cytoplasmic poly(A)⁺ RNA is about half that of total single-copy DNA. Similar results were obtained for cDNA made from unfertilized egg poly(A)⁺ RNA. In contrast, sequences expressed in gastrula nuclear RNA have diverged almost as much as total single-copy DNA.     

RNA synthesised during oogenesis and early embryogenesis in an insect egg (Euscelis plebejus)

Summary RNA labelled during oogenesis or early embryogenesis was isolated from eggs of the leaf hopperEuscelis plebejus. The polyadenylated RNA fraction deposited during early oogenesis accounted for approximately 2.7% of the total RNA content of the newly laid egg. This fraction differed significantly in molecular weight (15–32 S) from poly(A)-containing RNA synthesised between early cleavage and early germ anlage stages (4–20S). Locally injected³H-uridine spread through the egg within approximately 3 h. A considerable fraction (25–35%) of label injected as³H-uridine during early cleavage was recovered in DNA at subsequent stages (10–20 h later); labelled RNA was not found prior to the cellular blastoderm stage. When the yolk-endoplasm was separated from the blastoderm cells, only the latter contained demonstrable amounts of RNA synthesised by the embryo. Of the precursor incorporated into embryonic RNA, approximately 10% was found in the polyadenylated fraction at the early blastoderm stage, but only 3% at the early germ anlage stage. No differences in size distribution of polyadenylated RNA were evident between anterior and posterior halves of the early germ anlage stage.Supported by the Deutsche Forschungsgemeinschaft, SFB 46     

Sequences coding for proteins expressed in liver and for globin in poly(A)+ and poly(A)− RNA fractions from nuclei and cytoplasm of chicken immature red blood cells

By hybridization with [³H]labeled globin cDNA the contents of globin coding sequences in total nuclear RNA, poly(A)⁺nuclear RNA, poly(A)^--nuclear RNA and polysomal RNA of chicken immature red blood cells was determined to be 0.86%, 20%, 0.42% and 1% respectively. As the poly(A)⁺-fraction comprises only about 2% of total nuclear RNA, globin coding sequences are distributed with 49% in the poly(A)⁺-fraction and with 51% in the poly(A)^--fraction.Part of the mRNA sequences which are found in liver are also transcribed in immature red blood cells. These sequences are enriched in poly(A)⁺-nuclear RNA as the globin coding sequences but their total amount in the poly(A)⁺-fraction is much smaller than in the poly(A)^--fraction.When nuclear RNA from immature red blood cells was translated in an ascites tumor cell-free system, 20% of the newly synthesized proteins were globin chains. The percentage of globin chains in the newly synthesized proteins increased to over 70% when poly(A)⁺-nuclear RNA was translated. Only about 7.5% of globin chains were found in proteins coded by poly(A)^--nuclear RNA.     

Plant Desiccation and Protein Synthesis : V. Stability of Poly (A) and Poly (A) RNA during Desiccation and Their Synthesis upon Rehydration in the Desiccation-Tolerant Moss Tortula ruralis and the Intolerant Moss Cratoneuron filicinum

Upon desiccation of gametophytes of the desiccation-tolerant moss Tortula ruralis preexisting pools of poly(A)⁻ RNA (rRNA) remain inact, regardless of the speed at which desiccation is achieved. Preexisting poly(A)⁺ RNA pools (mRNA) are unaffected by slow desiccation but are substantially reduced during rapid desiccation. Poly(A)⁻ RNA involved in protein synthesis is also unaffected by desiccation, whereas the levels of polysomal poly(A)⁺ RNA in rapid- and slow-dried moss closely reflect the state of the protein synthetic complex in these dried samples.

Poly(A)⁻ RNA pools, both total and polysomal, are also stable during the rehydration of both rapid- and slow-dried moss. The total poly(A)⁺ RNA pool decreases upon rehydration, but this reduction is simply an expression of the normal turnover of poly(A)⁺ RNA in this moss. Analysis of polysomal fractions during rehydration reveals the continued use of conserved poly(A)⁺ RNA for protein synthesis. The rate of synthesis of poly(A)⁺ RNA upon rehydration appears to depend upon the speed at which prior desiccation is administered. Rapidly dried moss synthesizes poly(A)⁺ RNA at a faster rate, 60 to 120 minutes after the addition of water, than does rehydrated slowly dried moss. Recruitment of this RNA into the protein synthetic complex also follows this pattern. Comparative studies involving the aquatic moss Cratoneuron filicinum are used to gain an insight into the relevance of these findings with respect to the cellular mechanisms associated with desiccation tolerance.

     

Changes in the polyadenylated messenger RNA population during development of Dictyostelium discoideum

The program of gene expression during the life cycle of Dictyostelium discoideum has been assessed by molecular hybridization of cDNA probes with polysomal RNA extracted at the following different stages of development: vegetative growth, interphase (2.5 hr), aggregation (8 hr), postaggregation (12 hr), and preculmination (18 hr). Several different cDNA probes were used. Two probes were prepared from vegetative stage poly(A⁺) RNA, one representing all species present and the other enriched for abundant species. A third cDNA probe was prepared from preculmination stage polysomal RNA and a fourth probe consisted of the preculmination stage cDNA depleted in those species also present at the vegetative stage. Hybridization of the various probes with the different polysomal RNA preparations has revealed developmental changes in the mRNA populations. These changes were not detected in an aggregation less mutant under similar conditions of starvation. Abundant RNA species of vegetative cells were found to drop to low levels, especially during the aggregation period. Fifty percent by mass of the RNA present in polysomes at 18 hr is not present during vegetative growth. Some of the new RNA species appeared during interphase and the remaining during the postaggregation period. A gradual increase in the number of copies per cell of certain RNA species comprising both new species as well as some shared with vegetative cells was observed throughout development. Other results indicated that the composition of polysomal and cytoplasmic RNA is similar during vegetative growth but differs markedly at 18 hr of development. Also, cytoplasmic RNA at 18 hr contained, in addition to polysomal RNA, a large proportion by mass of nonpolysomal RNA similar to vegetative RNA. The number of polysomal RNA species detected by this analysis during vegetative growth and during the preculmination stage were estimated to be 3000 and 3700, respectively. The number of copies of these RNA species ranged between 30 and 2000 per cell during vegetative growth and 3 to 300 per cell in polysomes at 18 hr. Developmentally induced RNAs which were preferentially distributed among abundant and intermediate classes were estimated to number 700–900 species.     

Stored mRNA in cotyledons of Vigna unguiculata seeds: nucleotide sequence of cloned cDNA for a stored mRNA and induction of its synthesis by precocious germination

By differential hybridization screening, we previously selected a class of cDNA clones from a gt10 cDNA library that was constructed from the total poly(A)⁺ RNA of mature cowpea cotyledons (Plant Cell Physiol 31: 39–44, 1990). pSAS10, a clone of this class, hybridized with a cDNA probe complementary to poly(A)⁺ RNA from cotyledons collected 1 day after the onset of imbibition (DAI), but not with the cDNA probe from cotyledons at development stage II (13 to 15 days after flowering, DAF). pSAS10 mRNA was detectable only in cotyledons at development stage III (17 to 19 DAF) or later, and its level began to decline when seeds germinated. We have suggested that pSAS10 mRNA is likely to belong to the class of stored mRNA or the mRNA that is formed at the late stage of seed maturation, is conserved in quiescent seeds and becomes functional at the early stage of germination. We determined the nucleotide sequence of pSAS10 cDNA consisting of 459 bp and an approximately 36 bp poly(A) tract, and deduced the amino acid sequence of its product, a 10-kDa cysteine-rich polypeptide. Synthesis of pSAS10 mRNA was induced just before germination began, not only in mature seeds but also in immature seeds even at stages I (9 to 11 DAF) and II (13 to 15 DAF) if they were placed under conditions suitable for germination.