Polyadenylic acid-containing RNA in Xenopus laevis oocytes

The quantity of poly(A)-containing RNA is measured in Xenopus laevis oocytes as a function of developmental stage. The amount of poly(A)-containing RNA per oocyte, 0.7 to 1.0% of the total RNA, remains relatively constant from early vitellogenesis until ovulation. It is largely present in the cytoplasm of the oocyte in the form of a ribonucleoprotein complex. The poly(A) sequence is approximately 100 bases in length and is attached to molecules of heterogeneous sedimentation coefficients.     

Association of maternal and newly synthesized ribosomes with membranous noncytoskeletal structures in Xenopus laevis embryonic cells

In Xenopus laevis embryos a high concentration of both KCl and 0.5% DOC (sodium deoxycholate) is needed for maximal extraction of ribosomes and polysomes. We studied the nature of the structures that keep ribosomes and polysomes immobilized within the cytoplasm of embryonic cells at cleavage through tailbud stages, using various combinations of a low-salt buffer (20 mM KCl), a high-salt buffer (500 mM KCl), 0.5% DOC, and 0.5% Triton X-100. With a low-salt buffer and 0.5% DOC, but not Triton X-100, 80S ribosomal monomers and polysomes were liberated from the cytoplasmic rapidly sedimenting structures (RSS) to the soluble fraction. With a high-salt buffer (500 mM KCl), ribosomes were solubilized as 60S and 40S subunits together with about one-half of the total polysomes. When cells were homogenized in a low-salt buffer with added inhibitors of the cytoskeleton (cytochalasin B or colchicine), the majority of polysomes but not ribosomes were solubilized. These results provide evidence for the following conclusions. 1) Polysomes are bound to cytoskeletal structures in Xenopus embryos, but ribosomes, both maternal and newly synthesized, are associated with membranous noncytoskeletal structures. 2) The membranous structures consist of two compartments, one high-salt sensitive and the other high-salt resistant. 3) Ribosomes of the high-salt resistant group increase in amount with developmental stage and appear to be the precursor to the ribosomes of the high-salt sensitive group.     

Polyadenylic acid sequences in the RNA of Hyphomicrobium

Heterogeneous RNA containing polyadenylic acid [poly(A)] sequence has been isolated from Hyphomicrobium by affinity chromatography on oligothymidylic acid cellulose and polyuridylic acid Sepharose columns. About 0.1 to 0.3% of [3H]adenine-labeled RNA over a 60-min period is associated with poly(A) sequences. This percentage decreases to about 0.03 in a 20-h labeling period. The poly(A) tracts recovered after digestion with ribonuclease A and T1 are composed of greater than 95% adenine residues and are up to 200 nucleotides in length with a predominant range of 15 to 40 nucleotides. Adenosine and AMP are present in the ratio of 1:36 in alkaline digests of Hyphomicrobium poly(A) tracts. This is compatible with nucleotide lengths determined on acrylamide gels and location at the 3'-OH terminus of the RNA molecule.     

Polyadenylic acid sequences in rhinovirus RNA species from infected human diploid cells.

Polyadenylic acid sequences were shown to be present in rhinovirus virion RNA. Virus-specified RNA from human embryo lung cells infected with rhinovirus also contained polyadenylic acid but did not contain any polyuridylic acid sequences.     

The synthesis of polyadenylic acid-containing ribonucleic acid by isolated mitochondria from Ehrlich ascites cells.

The synthesis of poly(A)-containing RNA by isolated mitochondria from Ehrlich ascites cells was studied. Isolated mitochondria incorporate [3H]AMP or [3H]UTP into an RNA species that adsorbs on oligo (dT)-cellulose columns or Millipore filters. Hydrolysis of the poly(A)-containing RNA with pancreatic and T1 ribonucleases released a poly(A) sequence that had an electrophoretic mobility slightly faster than 4SE. In comparison, ascites-cell cytosolic poly(A)-containing RNA had a poly(A) tail that had an electrophoretic mobility of about 7SE. Sensitivity of the incorporation of [3H]AMP into poly(A)-containing RNA to ethidium bromide and to atractyloside and lack of sensitivity to immobilized ribonuclease added to the mitochondria after incubation indicated that the site of incorporation was mitochondrial. The poly(A)-containing RNA sedimented with a peak of about 18S, with much material of higher s value. After denaturation at 70 degrees C for 5 min the poly(A)-containing RNA separated into two components of 12S and 16S on a 5-20% (w/v) sucrose density gradient at 4 degrees C, or at 4 degrees and 25 degrees C in the presence of formaldehyde. Poly(A)-containing RNA synthesized in the presence of ethidium bromide sedimented at 5-10S in a 15-33% (w/v) sucrose density gradient at 24 degrees C. The poly(A) tail of this RNA was smaller than that synthesized in the absence of ethidium bromide. The size of the poly(A)-containing RNA (approx. 1300 nucleotides) is about the length necessary for that of mRNA species for the products of mitochondrial protein synthesis observed by ourselves and others.     

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.     

Short polyadenylic acid sequences in insect chorion messenger RNA

mRNAs for silkmoth chorion proteins contain polyadenylic acid sequences that comprise approximately 11–14% of the adenines and 3.5–5.2% of the phosphates. The poly(A) fragments recovered from these mRNAs have an average length of 28–39 (weight average) or 19–34 (number average) residues, after an in vivo labeling period of 24 hr. Short poly(A) fragments, of only slightly larger size, were observed even with labeling periods as brief as 3 hr.     

RNA synthesis in nuclei isolated from early embryos of Xenopus laevis.

1. Rates of RNA synthesis in isolated Xenopus embryo nuclei decrease from blastula through gastrula and neurula stages to hatching tadpoles. 2. In blastula and gastrula nuclei, net synthesis of RNA continues for over 30 min, both in the presence of KCl at 0.4 M and in its absence. In nuclei from later stages, net synthesis continues for only about 10 min in the absence of KCl. 3. At low ionic strength, RNA synthesis in all nuclei is greater with optimum Mg-2+ (6 mM) than with optimum Mn-2+ (1 mM). At high ionic strength the reverse is true. 4. An unusual feature, which gradually disappears as development proceeds, is that curves relating RNA synthesis to KCl concentration show a peak at 0.1 M KCl. In blastula nuclei, RNA synthesis is more rapid at 0.1 M KCl than at 0.4 M. 5. This peak at low ionic strength is not observed in the presence of the initiation inhibitor rifamycin AF/013. It is concluded that the peak arises from initiation of RNA synthesis by an excess of RNA polymerases bound non-specifically to the isolated nuclei. The residual synthesis, representing elongation of chains that were initiated in vivo, still declines as development progresses. 6. In blastula nuclei, over half of the RNA synthesis is effected by polymerase II (inhibited by alpha-amanitin), the proportion remaining roughly constant with increasing ionic strength. In neurula nuclei, the proportion rises from about one-half to three-quarters. The initiation-dependent peak in blastula and gastrula nuclei is contributed by both alpha-amanitin-sensitive and alpha-amanitin-resistant enzymes.     

The nucleotide sequences of 5.8-S ribosomal RNA from Xenopus laevis and Xenopus borealis

1. The nucleotide sequence of 5.8-S rRNA from Xenopus laevis is given; it differs by a C in equilibrium U transition at position 140 from the 5.8-S rRNA of Xenopus borealis. 2. The sequence contains two completely modified and two partially modified residues. 3. Three different 5' nucleotides are found: pU-C-G (0.4) pC-G (0.2) and pG (0.4). 4. The 3' terminus is C not U as in all other 5.8-S sequences so far determined. 5. The X. laevis sequence differs from the mammalian and turtle sequences by five and six residue changes respectively. 6. A ribonuclease-resistant hairpin loop is a principle feature of secondary structure models proposed for this molecule. 7. Sequence heterogeneity may occur at one position at a very low level (approximately 0.01) in X. laevis 5.8-S rRNA, while none was detected in X. borealis or HeLa cell 5.8-S rRNA.     

A possible maternal-effect mutant of Xenopus laevis: II. Studies of RNA synthesis in dissociated embryonic cells

Embryos from a female of Xenopus laevis (designated as no. 65) arrest development at gastrulation and are assumed to be ova-deficient mutant. We dissociated these embryos and studied RNA synthesis at different stages. The cells from the ova-deficient embryos reaggregated quite actively as wild-type embryo cells until the late gastrula stage. RNA synthesis was normal at the early blastula stage but greatly inhibited by the late blastula (stage 9.5) stage, when the synthesis of DNA and protein was still not inhibited appreciably. Thus, inhibition in RNA synthesis appears to be the first manifestation of the maternal defect that occurs before the gastrulation arrest.