Preribosomal RNA processing in Xenopus oocytes does not include cleavage within the external transcribed spacer as an early step.

Recently it has been reported that U3 snRNA is necessary for: (a) internal cleavage at +651/+657 within the external transcribed spacer (ETS) of mouse precursor ribosomal RNA (pre-rRNA); and (b) cleavage at the 5' end of 5.8S rRNA in Xenopus oocytes. To study if U3 snRNA plays a role at more than one processing site in the same system, we have investigated whether internal cleavage sites exist within the ETS of Xenopus oocyte pre-rRNA. The ETS of Xenopus pre-rRNA contains the consensus sequence for the mammalian early processing site (+651/+657 in mouse pre-rRNA), but freshly prepared RNA from Xenopus oocytes has no cuts in this region. The only putative cleavage sites we found in the ETS of Xenopus oocyte pre-rRNA are a cluster further downstream of the mouse early processing site consensus sequence. This cluster is not homologous to the mouse +651/+657 sites because unlike the latter it is (a) not abolished by disruption of U3 snRNA, (b) not cleaved during early steps of pre-rRNA processing, and (c) lacks sequence similarity to the +651/+657 consensus. Therefore, pre-rRNA of Xenopus oocytes does not cleave within the ETS as an early step in rRNA processing. We conclude that cleavage within the ETS is not an obligatory early step needed for the rest of rRNA maturation.     

Molecular characterization of DNA puff II/9A genes in Sciara coprophila

A cDNA clone, pSDII/9, that hybridizes in situ to ecdysone-regulated DNA puff II/9A in Sciara coprophila was used as a probe to isolate a Sciara genomic clone. lambda pSDII/9, which contains a 14.7 x 10(3) base-pair DNA insert. The full-length cDNA insert was sequenced and mapped to gene II/9-1 on the genomic clone. A second gene (II/9-2), transcribed in the same direction as II/9-1, was also mapped to lambda pSDII/9, and its nucleic acid sequence was found to be 85% similar to that of gene II/9-1. An RNase protection assay demonstrates that gene II/9-1 contains a single intron that also exists in gene II/9-2 according to sequencing analysis and primer extensions of RNA encoded by this gene. Computer analyses of the deduced amino acid sequences of genes II/9-1 and II/9-2 indicate that the two DNA puff-encoded proteins are mostly alpha-helical coiled-coils. The 5'-flanking sequences of both genes contain regions that are similar to other ecdysone-regulated genes from Drosophila melanogaster.     

Sequence analysis of 28S ribosomal DNA from the amphibian Xenopus laevis.

We have determined the complete nucleotide sequence of Xenopus laevis 28S rDNA (4110 bp). In order to locate evolutionarily conserved regions within rDNA, we compared the Xenopus 28S sequence to homologous rDNA sequences from yeast, Physarum, and E. coli. Numerous regions of sequence homology are dispersed throughout the entire length of rDNA from all four organisms. These conserved regions have a higher A + T base composition than the remainder of the rDNA. The Xenopus 28S rDNA has nine major areas of sequence inserted when compared to E. coli 23S rDNA. The total base composition of these inserts in Xenopus is 83% G + C, and is generally responsible for the high (66%) G + C content of Xenopus 28S rDNA as a whole. Although the length of the inserted sequences varies, the inserts are found in the same relative positions in yeast 26S, Physarum 26S, and Xenopus 28S rDNAs. In one insert there are 25 bases completely conserved between the various eukaryotes, suggesting that this area is important for eukaryotic ribosomes. The other inserts differ in sequence between species and may or may not play a functional role.     

Fine structure of ribosomal RNA. II. Distribution of methylated sequences within Xenopus laevis rRNA.

The distribution of methyl groups in rRNA from Xenopus laevis was analyzed by hybridization of rRNA to subfragments of either of two cloned rDNA fragments, X1r11 and X1r12, which together constitute a complete rDNA repeat unit. Using a mixture of 3H-methyl plus 32P-labelled rRNA as probe, the molar yield of methyl groups per rRNA region in hybrid could be calculated. For this calculation the length of the rRNA coding region in each DNA subfragment is needed, which was determined for X1r11 subfragments by the nuclease S1 mapping method of Berk and Sharp. The results show that both in 18S and 28S rRNA the methyl groups are nonrandomly distributed. For 18S rRNA, clustering was found within a 3' terminal fragment of 310 nucleotides. For 28S rRNA, clustering of methyl groups was found within a region of 750 nucleotides in length, which ends 500 nucleotides from the 3' end. In contrast, the 28S rRNA 5' terminal region of 900 nucleotides is clearly undermethylated. The general position of methyl groups in 28S rRNA correlates with the location of evolutionarily conserved sequences in this molecule, as recently determined in our laboratory.     

U3 snoRNA may recycle through different compartments of the nucleolus

A model is proposed in which U3 small nucleolar RNA (snoRNA) is recruited from an inactive, stored form in the dense fibrillar component (DFC) of the nucleolus to an active form that is associated with the initial ribosomal RNA (rRNA) precursor. The initial steps of rRNA processing occur in the DFC, and then it is proposed that the U3 snoRNA moves with intermediates in rRNA processing from the DFC to the granular component (GC) of the nucleolus. The nucleolar protein fibrillarin is located primarily in the DFC, and it is suggested that the complex of fibrillarin and U3 snoRNA dissociates when U3 snoRNA transits to the GC. Finally, when U3 snoRNA is released from the processed rRNA, the tether holding the rRNA in the nucleolus is broken and rRNA can then be exported from the nucleolus to the cytoplasm. U3 snoRNA is hypothesized to recycle back from the GC to the DFC where it is stored until future association with another initial rRNA precursor. Data supporting this model are summarized. U3 snoRNA is also stored in the coiled body of interphase cells and in the nucleolar remnants and prenucleolar bodies of mitotic cells, and there may be some similarity in the binding sites for stored U3 snoRNA in the DFC and in these structures. Received: 16 September 1996 / Accepted: 11 November 1996     

Effect of Fish Oil Diet on Fatty Acid Composition of Phospholipids of Brain Membranes and on Kinetic Properties of Na+, K+-ATPase Isoenzymes of Weaned and Adult Rats

Abstract: The influence of dietary (n-3) fatty acids (such as eicosapentaenoic and docosahexaenoic acids) as found in fish oil on Na⁺ sensitivity and ouabain affinity of Na⁺, K⁺-ATPase isoenzymes (α1, α2, α3) was studied in whole brain membranes from weaned and adult rats fed diets for two generations. The long chain (n-3) fatty acids supplied by fish oil decreased the fatty acids of the (n-6) series compared with the standard diet, resulting in a decrease in the (n-6)/(n-3) molar ratio in both 21 - and 60-day- old rats. On the basis of ouabain titration, three inhibitory processes with markedly different affinities were associated with isoenzymes, i.e., low affinity (α1), high affinity (α2), and very high affinity (α3). It appears that the fish oil diet, in part via the modification of membrane fatty acid composition, altered the proportion and ouabain affinity of isoenzymes. Na⁺ sensitivity is the best criterion of physiologic change induced by fish oil diet. We calculated the Na⁺ activation for each isoenzyme and found one Na⁺ sensitivity and two Na⁺ sensitivities per isoenzyme in weanling and adult rats fed different diets, respectively. In contrast to α2 and α3, α1 appears insensitive to membrane change induced by fish oil diet. Fish oil diet, which is known to confer cardioprotection, induced significant modulation of Na⁺, K⁺-ATPase isoenzymes at the brain level.