Structural gene sets active in embryos and adult tissues of the sea urchin

Structural gene sequences active in a variety of sea urchin adult and embryo tissues are compared. A single-copy ³H-DNA fraction, termed mDNA, was isolated, which contains sequences complementary to the messenger RNA present on gastrula stage polysomes. Gastrula message sequences are 50 fold concentrated in the mDNA compared to total single-copy DNA. mDNA reactions were carried out with excess mRNA from blastula, pluteus, exogastrula, adult ovary, tubefoot, intestine, and coelomocytes, and with excess total mature oocyte RNA. A single-copy ³H-DNA fraction totally devoid of gastrula message sequences, termed null mDNA, was also reacted with these RNAs. Large differences in the extent of both mDNA and null mDNA reaction with the various RNAs were observed, indicating that in each state of differention a distinct set of structural genes is active, generally characterized by several thousand specific sequences. The complexity of gastrula mRNA was shown in previous work to be about 17 × 10⁶ nucleotides. In units of 10⁶ nucleotides, the complexities of the RNA sequence reacting with mDNA and with null mDNA in each tissue are, respectively, as follows: intestine mRNA; 2.1 and 3.7; coelomocyte mRNA: 3.5 and ≤1.4; tubefoot mRNA: 2.7 and ≤0.4; ovary mRNA: 13 and 6.7; oocyte total RNA: 17 and 20; blastula mRNA: 12 and 15; pluteus mRNA: 14 and ≤0.6; exogastrula mRNA: 14 and ≤0.6. The total complexity of each mRNA population is the sum of these values, as verified for several cases by reactions with total single-copy DNA. A relatively small set of mRNAs, the complexity of which is about 2.1 × 10⁶ nucleotides, appears to be shared by several of the tissues studied.     

Effects of estrogen on gene expression in rooster liver.

The effects of estrogen on RNA sequence complexity and sequence frequency were studied in rooster liver. Both control and estrogen-treated liver contained total RNA sequence diversity of approximately 4.2 × 10⁷ nucleotides. Two components were found in the reaction of chicken liver or brain RNA with unique DNA: RNA species present at high concentration and RNA species about 100-fold less abundant. Approximately 7 × 10⁶ nucleotides of RNA sequence complexity were present at high concentration in estrogen-treated liver but not at high concentration in control liver.     

Differential accumulation and localization of maternal poly(A)-containing RNA during early development of the ascidian,Styela

The regional distribution of poly(A)⁺ RNA was examined in sections of Styela oocytes and fertilized eggs by in situ hybridization with [³H]poly(U). The nucleus and cytoplasm of previtellogenic oocytes contain equivalent densities of [³H]poly(U) binding sites. The concentration of these sites is reduced in the cytoplasm, but not the nucleus, during vitellogenesis. Consequently, the germinal vesicle (GV) plasm of mature oocytes is characterized by an eightfold elevation in [³H]poly(U) binding activity relative to the surrounding cytoplasm. The distinctive cytoplasmic regions of the mature oocyte do not exhibit differential concentrations of [³H]poly(U) binding sites. Following fertilization which triggers GV breakdown, meiosis, and ooplasmic segregation, the high density of [³H]poly(U) binding sites characteristic of the GV plasm is conserved in the basophilic cytoplasm during its extensive migration and eventual accumulation in the animal hemisphere of the egg. The insensitivity of the [³H]poly(U) binding sites of the basophilic cytoplasm to actinomycin D suggests that they are of maternal origin. It is concluded that maternal poly(A)⁺ RNA is subject to differential accumulation in the GV plasm and its derivative ooplasm during the early development of Styela.     

Regulation of histone synthesis during early Urechis caupo (Echiura) development

The histones present in mature oocytes and embryos of Urechis caupo and their pattern of synthesis during early development have been characterized. Acid-soluble proteins extracted from mature oocyte germinal vesicles and from embryonic nuclei were analyzed by two-dimensional polyacrylamide gel electrophoresis. Histones are accumulated in the mature oocytes in amounts sufficient to provide for the assembly of chromatin through the 32- to 64-cell stage of embryogenesis. Two H1 histones, which appear to be variants, were found. Germinal vesicles and cleavage-stage nuclei are enriched in H1M (maternal). During late cleavage a faster-migrating H1, H1E (embryonic), appears among the nuclear histones and, as embryogenesis continues, replaces H1M as the predominant H1. No new core histone variants are detected during early development. Examination of [³H]lysine-labeled histones from germinal vesicles and embryonic nuclei reveals stage-specific patterns of histone synthesis. H1M is the major H1 species synthesized in mature oocytes. After fertilization, a switch to the predominant synthesis of H1E occurs. Comparison of the [³H]lysine incorporated into H1E and core histones indicates that H1E synthesis is disproportionately high from midcleavage through the midblastula stage. By the gastrula stage, a balanced synthesis of H1E and each core histone is established. The results indicate that there is noncoordinate regulation of H1 and core histone synthesis during Urechis development.     

Gene expression during development of Blastocladiella emersonii

To evaluate gene expression during sporulation and early development of the aquatic fungus Blastocladiella emersonii, the nucleotide sequence complexity of the polysomal RNA has been measured at different stages. To assess the effect of medium composition on gene expression, similar experiments were completed during early development in a range of simple to complex media. The polysomal RNA sequence complexity was measured by hybridization with single-copy tracer DNA and with a complex class-enriched cDNA fraction copied from the stored zoospore poly(A⁺)RNA. Forty-four to eighty-six percent (8.2 × 10⁶ to 16 × 10⁶ nucleotides) of the single-copy DNA sequence complexity was found on polysomes, depending upon the stage examined or the medium used, compared to 42.5% (8 × 10⁶ nucleotides) in the stored RNA pool of zoospores. The highest levels of complexity occurred during the two periods of active differentiation, sporulation and germination. During starvation-induced sporulation, and average of 82% of the total asymmetrically transcribed complexity was expressed; half of this complexity was lost prior to the completion of zoospore differentiation and was missing from the zoospore-stored RNA pool. During the first 30 min of zoospore germination the level of sequence complexity increased by 46 to 66% over the zoospore level, depending upon the medium used. The polysomal RNA complexity then decreased by a nearly equal amount between 30 and 60 min when the cells entered the growth phase. An inverse relationship was found between the richness of the medium and the level of sequence complexity found on polysomes. The data indicate that sequences representative of most of the zoospore-stored poly(A+)RNA were expressed at all other stages and maintained by turnover and resynthesis. In addition, significant numbers of new sequences were also expressed, particularly during stages of active differentiation. Cells that germinated and completed early development in an inorganic starvation medium showed a marked loss of the middle and high abundance classes of poly(A⁺)RNA and slight enrichment for the low abundance class.     

Differential accumulation of two size classes of poly(A) associated with messenger RNA during oogenesis in Xenopus laevis

The RNA of full-grown oocytes of Xenopus laevis contains two distinct size classes of poly(A), designated poly(A)_S and poly(A)_L, which contain 15–30 (mean = 20) and 40–80 (mean = 61) A residues, respectively. Both poly(A)_L and poly(A)_S are associated with RNA which is heterogeneous in size. The two classes of poly(A)⁺ RNA can be separated by affinity chromatography: Only poly(A)_L⁺ RNA binds to oligo(dT)-cellulose under appropriate conditions, but up to 50% of the poly(A)_S⁺ RNA can be isolated from the void fraction by binding to poly(U)-Sepharose. Both classes of poly(A)⁺ RNA are active as messenger RNA in an in vitro system and yield identical patterns of in vitro protein products. Previtellogenic oocytes contain almost exclusively poly(A)_L, which accumulates up to vitellogenesis but remains almost constant in amount (molecules/oocyte) during vitellogenesis and in the full-grown oocyte. Poly(A)_S accumulates (molecules/oocyte) from early vitellogenesis up to the full-grown oocyte. The total number of poly(A)⁺ RNA molecules per oocyte increases throughout oogenesis from 2 × 10¹⁰/previtellogenic oocyte [80–90% poly(A)_L] to 20 × 10¹⁰/full-grown oocyte (25–40% poly(A)_L). It is argued that poly(A)_S is protected from degradation in the oocyte, thus stabilizing the “maternal” poly(A)⁺ mRNA.     

Precursors of chloroplast ribosomal RNA in Euglena gracilis

Incorporation of ³²P into mature chloroplast rRNA species of MW 1.1 × 101 and 0.56 × 10⁶ has been followed in Euglena gracilis by pulse and pulse chase experiments. Mature rRNA species have precursors of MW 1.16 × 10⁶ ± 0.01 × 10⁶ and 0.64 × 106 ± 0.01 × 10⁶ resp. These precursors have base composition and hydridization properties similar to those of the mature, rRNA species. No evidence of a single common precursor to these molecules was found. Rifampicin did not affect the synthesis of chloroplast rRNA.     

Quantitative changes in total RNA, total poly(A), and ribosomes in early mouse embryos

To obtain information on the amounts and major classes of RNA stored in the mouse egg and accumulated during cleavage, we determined the contents of total RNA, total poly(A), and ribosomes from the 1-cell stage to blastocyst. Using purified RNA for assay, we obtained an RNA content of 0.35 ng in the unfertilized egg, 0.24 ng in 2-cell, 0.69 ng in 8- to 16-cell, and 1.47 ng in early bastocyst (32 cells). As derived from EM morphometry, the number of ribosomes accounts for 60–70% of the total RNA content at all these stages; the marked increase in ribosomal number during cleavage is attributable entirely to new synthesis. Hybridization with [³H]poly(U) in solution yielded a poly(A) content of 0.7 pg for the unfertilized egg and 0.83 pg for the 1-cell embryo. The poly(A) content dropped sharply, to 0.26 pg per embryo, by the late 2-cell stage and increased to 0.44 pg in 8- to 16-cell embryos and 1.42 pg in early blastocysts. Hybridization in situ gave a similar pattern and also revealed a heavy labeling of embryo nuclei from the 2-cell onward but very little, if any, labeling of the pronuclei of 1-cell embryos, suggesting an absence, or low level, of poly(A)+ RNA synthesis at the 1-cell but an active synthesis at the 2-cell and later stages. These findings and other available evidence(e.g., R. Bachvarova and V. De Leon, 1980, Develop. Biol.74, 1–8) suggest that the mouse embryo inherits a large supply of maternal mRNA but that the bulk of this RNA is eliminated in the 2-cell embryo. In situ hybridization was used to study the relative concentration of poly(A) in ovarian oocytes. In growing oocytes, the cytoplasmic concentration of poly(A) remains about the same, suggesting that the accumulation of poly(A)+ RNA is proportional to oocyte growth. The poly(A) content declines about twofold between the time of completion of oocyte growth and fertilization. The germinal vesicle continues to be labeled up to the time of ovulation, raising the possibility that poly(A)+ RNA synthesis (and presumably turnover) occurs in fully grown oocytes.     

Localization of nucleic acids in the nucleoli of oocytes and early embryos of mouse and hamster: An autoradiographic study

Mouse preovulatory oocytes, zygotes, parthenogenetically activated pronuclear oocytes, and early embryos, as well as hamster zygotes, were analyzed, by autoradiography, for the distribution of either “maternal” or newly synthesized RNAs. Early mouse embryos were also examined for the distribution of newly replicated DNA. Special attention was attributed to NLBs in oocytes or to NPBs in early embryos. In mouse oocytes, [5-³H]uridine radioactivity accumulated (after a 2-hr pulse) in vitro, in addition to other nuclear compartments, in the central compact material of the NLBs. There was no cytoplasmic labeling. In all parthenogenetic pronuclear embryos developed from similarly labeled oocytes, this label was distinctly detectable in the central compact material of the NPBs; less intensive labeling was seen in the nucleoplasm and cytoplasm. On the contrary, the central compact part of the mouse NPB did not show labeling in DNA after a continuous culture with [6-³H]thymidine. In mouse and hamster pronuclear zygotes, convincing evidence was obtained for a lack of any newly synthesized nucleic acids in the compact material of NPBs using 4- to 10-hr culture with [8-³H]adenosine. Based on these data, it was shown that the NLBs of oocytes or NPBs of early embryos probably contain RNAs synthesized during the last stages of antral follicle oocyte differentiation. This unique pathway of RNAs in the oocyte—embryo system may explain the specific morphology of both oocyte and early embryo “nucleoli.” © 1995 Wiley-Liss, Inc.     

Endonucleolytic cleavage of murine leukemia virus 35S RNA by microsome-associated nuclease.

Moloney murine leukemia virus 35S RNA (molecular weight 3 to 3.4 × 10⁶) is cleaved by nuclease activity present in microsomal fractions from MLV infected or uninfected mouse embryo cells to two RNA species of approximate molecular weights 1.8 × 10⁶ and 1.5 × 10⁶. Microsomal fractions from MLV infected and uninfected cells also contained nucleolytic activity that solubilized [³H]poly(A)·poly(U) but not [³H]poly(C) or [³H]poly(U); the cleavage of poly(A)·poly(U) was inhibited by ethidium bromide. The cleavage of MLV RNA was also inhibited by ethidium bromide, suggesting double stranded regions in 35S RNA as the site of cleavage.     

Poly(A) and synthesis of polyadenylated RNA in the preimplantation mouse embryo

Investigations were conducted to quantitate polyadenylic acid and estimate the synthesis of polyadenylated RNA in mouse embryos at several stages of preimplantation development. Poly(A) was assayed by molecular hybridization of total embryonic RNA with [³H]polyuridylic acid. The mean values of poly(A) in the ovulated oocytes and in the one-cell, two-cell, and blastocyst stages of the embryo were 1.9, 1.6, 0.68, and 3.8 pg, respectively. Synthesis of polyadenylated RNA was estimated by affinity chromatography of [³H]uridine-labeled embryo RNA on oligo(dT)-cellulose. The proportions of newly synthesized RNA bound by oligo(dT)-cellulose at the 2-cell, 8- to 16-cell, and blastocyst stages were 6.7, 3.5, and 3.3%, respectively. These results suggest that significant quantities of maternal mRNA are present during early development of the mouse, but that polyadenylation of RNA transcribed from the embryonic genome occurs as early as the two-cell stage.