Electron microscopic visualization of sites of nascent DNA synthesis by streptavidin-gold binding to biotinylated nucleotides incorporated in vivo

Biotinylated nucleotides (bio-11-dCTP, bio-11-dUTP, and bio-7-dATP) were microinjected into unfertilized and fertilized Xenopus laevis eggs. The amounts introduced were comparable to in vivo deoxy-nucleoside triphosphate pools. At various times after microinjection, DNA was extracted from eggs or embryos and subjected to electrophoresis on agarose gels. Newly synthesized biotinylated DNA was analyzed by Southern transfer and visualized using either the BluGENE or Detek-hrp streptavidin-based nucleic acid detection systems. Quantitation of the amount of biotinylated DNA observed at various times showed that the microinjected biotinylated nucleotides were efficiently incorporated in vivo, both into replicating endogenous chromosomal DNA and into replicating microinjected exogenous plasmid DNA. At least one biotinylated nucleotide could be incorporated in vivo for every eight nucleotides of DNA synthesized. Control experiments also showed that heavily biotinylated DNA was not subjected to detectable DNA repair during early embryogenesis (for at least 5 h after activation of the eggs). The incorporated biotinylated nucleotides were visualized by electron microscopy by using streptavidin-colloidal gold or streptavidin-ferritin conjugates to bind specifically to the biotin groups projecting from the newly replicated DNA. The incorporated biotinylated nucleotides were thus made visible as electron-dense spots on the underlying DNA molecules. Biotinylated nucleotides separated by 20-50 bases could be resolved. We conclude that nascent DNA synthesized in vivo in Xenopus laevis eggs can be visualized efficiently and specifically using the techniques described.     

Initiation of replication at specific origins in DNA molecules microinjected into unfertilized eggs of the frog Xenopus laevis

Initiation of DNA replication at specific origins was observed by electron microscopy after microinjection of pXlr11, pXlr14 or Col E1 plasmid DNA molecules into unfertilized eggs of the frog, Xenopus laevis. These results are in apparent contradiction with published reports (Harland and Laskey, Cell 21, 761-771, 1980; Laskey and Harland, Cell 24, 283-284, 1981) that specific origin sites were not used in Xenopus laevis eggs. We suggest that eucaryotic origins exist that both increase the probability of replication of contiguous sequences and determine the site at which replication is most likely to begin.     

Somatostatin-immunoreactive cells in the gastro-entero-pancreatic endocrine system of Xenopus laevis

Somatostatin-like immunoreactivity has been demonstrated in cells of the gastro-entero-pancreatic endocrine system of Xenopus laevis (Amphibia) using peroxidase-anti-peroxidase immunohistochemistry. The identified cells of the gastro-intestinal tract correspond to the "Open-Paraneurons" of FUJITA (1974). Somatostatin-immunoreactive cells were found in the stomach (fundic and pyloric region), the duodenum and in the anterior ileum, but not in the colon. The dimensions of these 'Somatostatin-Open-Paraneurons' were measured: mean maximum height = 41.31 micron (S.D. = 10.87 micron), mean maximum breadth = 8.73 micron (S.D. = 2.31 micron). Frequency distributions of the maximum height and of the maximum breadth were processed by use of a computer. Somatostatin-immunoreactive cells in the pancreatic islets occupied about 15 to 25% of the total islets volume. These cells are "Closed-Paraneurons" (according to FUJITA 1974) with a mean diameter of 11.68 micron (S.D. = 2.61 micron).     

Xenopus borealis and Xenopus laevis 28S ribosomal DNA and the complete 40S ribosomal precursor RNA coding units of both species.

We have determined the nucleotide sequence of Xenopus borealis 28S ribosomal DNA (rDNA) and have revised the sequence of Xenopus laevis 28S rDNA (Ware et al., Nucl. Acids Res. 11, 7795-7817 (1983)). In the regions encoding the conserved structural core of 28S rRNA (2490 nucleotides) there are only four differences between the two species, each difference being a base substitution. In the variable regions, also called eukaryotic expansion segments (ca. 1630 nucleotides) there are some 61 differences, due to substitutions, mini-insertions and mini-deletions. Thus, evolutionary divergence in the variable regions has been at least 20-fold more rapid than in the conserved core. A search for intraspecies sequence variation has revealed minimal heterogeneity in X. laevis and none in X. borealis. At three out of four sites where heterogeneity was found in X. laevis (all in variable regions) the minority variant corresponded to the standard form in X. borealis. Intraspecies heterogeneity and interspecies divergence in the 28S variable regions are much less extensive than in the transcribed spacers. The 28S sequences are from the same clones that were used previously for sequencing the 18S genes and transcribed spacers. The complete sequences of the 40S precursor regions of the two reference clones are given.     

Identification of the locations of the methyl groups in 18 S ribosomal RNA from Xenopus laevis and man

The 18 S ribosomal RNA from a variety of vertebrate species contains some 40 to 47 methyl groups. The majority of these are 2'-O-ribose substituents; the remaining few are on bases. Several lines of evidence have permitted the identification of the precise locations of the methyl groups in the primary structure of 18 S ribosomal RNA of Xenopus laevis and man. Digestion of RNA with T1 ribonuclease, followed by analysis of the methylated oligonucleotides yielded data on sequences immediately surrounding the methyl groups. Preparative hybridization of X. laevis 18 S ribosomal RNA restriction fragments of ribosomal DNA, followed by fingerprinting analysis on RNA recovered from the hybrids, allowed each methylated oligonucleotide to be mapped to a specific region within 18 S ribosomal RNA. The data on RNA oligonucleotides were correlated with Xenopus ribosomal DNA sequence data in the regions defined by the mapping experiments to identify the precise locations of most of the methyl groups in the X. laevis 18 S RNA sequence. The remaining uncertainties in Xenopus were solved with the aid of data from ribonuclease A fingerprints and, in a few instances, relevant oligonucleotide or sequence data from other laboratories. The locations of most of the methyl groups in human 18 S ribosomal RNA were deduced from the high degree of correspondence between methylated oligonucleotides from human and X. laevis 18 S RNA, together with knowledge of the human 18 S ribosomal DNA sequence. The remaining methylation sites in human 18 S RNA were located with assistance from relevant published comparative data. In the aligned sequences, human and other mammalian 18 S RNA are methylated at all the same positions as in X. laevis, and there are seven additional 2'-O-methylation sites in mammalian 18 S RNA. Further features of the methyl group distribution are briefly reviewed.     

The structure of rat 28S ribosomal ribonucleic acid inferred from the sequence of nucleotides in a gene.

The nucleotide sequence of a rat 28S rRNA gene was determined. The 28S rRNA encoded in the gene contains 4718 nucleotides and the molecular weight estimated from the sequence is 1.53 x 10(6). The guanine and cytosine content is 67%. The sequence of rat 28S rRNA diverges appreciably from that of Saccharomyces carlsbergensis 26S rRNA (about 50% identity), but more closely approximates that of Xenopus laevis 28S rRNA (about 75% identity). Rat 28S rRNA is larger than the analogous nucleic acids from yeast (3393 nucleotides) and X, laevis (4110 nucleotides) ribosomes. The additional bases are inserted in specific regions and tend to be rich in guanine and cytosine. 5.8S rRNA can interact with 28S rRNA by extensive hydrogen bonding at two sites near the 5' end of the latter.     

Effect of ouabain on the meiotic maturation of stage IV-V Xenopus laevis oocytes

Full-grown stage VI Xenopus laevis oocytes (1,200 to 1,300 micron) respond to progesterone stimulation by undergoing a series of physiological and morphological changes that are referred to as meiotic maturation. Oocytes in earlier stages of oogenesis (I through V) do not undergo these changes and remain in prophase arrest when exposed to this steroid. We have found that oocytes ranging from 850 micron (stage IV) to 1,000 micron (stage V) are capable of responding to progesterone under the appropriate conditions. Oocytes greater than or equal to 850 micron in diameter underwent germinal vesicle breakdown (GVBD) after 10-12 hr of exposure to progesterone when ouabain was added to the medium at a concentration greater than 2.5 X 10(-6) M. Under this culture condition, progesterone was now able to induce a 0.3- to 0.4-unit increase in the intracellular pH of stage IV-V oocytes, a 4- to 5-fold increase in 40s ribosomal protein S-6 phosphorylation, and a 2.3-fold increase in their rate of protein synthesis. All of these physiological changes are characteristic of full-grown stage VI oocytes undergoing meiotic maturation. In addition, we have found that oocytes greater than or equal to 750 micron are capable of amplifying maturation promoting factor (MPF) in their cytoplasm leading to GVBD. Therefore, stage IV-V Xenopus oocytes have the potential for undergoing meiotic maturation, but they are blocked at a point in prophase that appears to be alleviated by the combination of progesterone and ouabain.     

Isolation and organization of calf ribosomal DNA.

Ribosomal DNA (rDNA) from calf was isolated by three density gradient centrifugations. The first centrifugation in Cs2S04/BAMD was used to obtain partially resolved dG+dC-rich fractions from total DNA. The second and third centrifugations, in Cs2S04/Ag+, led to the isolation of an rDNA fraction characterized by a symmetrical band in CsCl, p = 1.724 g/cm3. This new procedure appears to be generally suitable for the isolation of rDNA and other dG+dC-rich repeated genes. The organization of isolated calf rDNA has been studied by restriction enzyme digestion and by hybridization with cloned rDNA from Xenopus laevis. The repeat unit of calf rDNA has a molecular weight of 21x10(6) and is split by EcoR1 into two fragments, 16x10(6) and 5.0x10(6), and by BamHI into seven fragments. EcoRI and BamHI sites have been mapped. Most of the 18S and 28S RNA genes and the transcribed spacer are contained in the small EcoRI fragment, while the non-transcribed spacer is localized in the large EcoRI fragment. This spacer showed length heterogeneity within a single individual; such heterogeneity is limited to two regions of the spacer.     

Human 18 S ribosomal RNA sequence inferred from DNA sequence. Variations in 18 S sequences and secondary modification patterns between vertebrates.

We have determined the DNA sequences encoding 18 S ribosomal RNA in man and in the frog, Xenopus borealis. We have also corrected the Xenopus laevis 18 S sequence: an A residue follows G-684 in the sequence. These and other available data provide a number of representative examples of variation in primary structure and secondary modification of 18 S ribosomal RNA between different groups of vertebrates. First, Xenopus laevis and Xenopus borealis 18 S ribosomal genes differ from each other by only two base substitutions, and we have found no evidence of intraspecies heterogeneity within the 18 S ribosomal DNA of Xenopus (in contrast to the Xenopus transcribed spacers). Second, the human 18 S sequence differs from that of Xenopus by approx. 6.5%. About 4% of the differences are single base changes; the remainder comprise insertions in the human sequence and other changes affecting several nucleotides. Most of these more extensive changes are clustered in a relatively short region between nucleotides 190 and 280 in the human sequence. Third, the human 18 S sequence differs from non-primate mammalian sequences by only about 1%. Fourth, nearly all of the 47 methyl groups in mammalian 18 S ribosomal RNA can be located in the sequence. The methyl group distribution corresponds closely to that in Xenopus, but there are several extra methyl groups in mammalian 18 S ribosomal RNA. Finally, minor revisions are made to the estimated numbers of pseudouridines in human and Xenopus 18 S ribosomal RNA.     

Oocyte and somatic 5S ribosomal RNA and 5S RNA encoding genes in Xenopus tropicalis.

We have investigated the structure of oocyte and somatic 5S ribosomal RNA and of 5S RNA encoding genes in Xenopus tropicalis. The sequences of the two 5S RNA families differ in four positions, but only one of these substitutions, a C to U transition in position 79 within the internal control region of the corresponding 5S RNA encoding genes, is a distinguishing characteristic of all Xenopus somatic and oocyte 5S RNAs characterized to date, including those from Xenopus laevis and Xenopus borealis. 5S RNA genes in Xenopus tropicalis are organized in clusters of multiple repeats of a 264 base pair unit; the structural and functional organization of the Xenopus tropicalis oocyte 5S gene is similar to the somatic but distinct from the oocyte 5S DNA in Xenopus laevis and Xenopus borealis. A comparative sequence analysis reveals the presence of a strictly conserved pentamer motif AAAGT in the 5'-flanking region of Xenopus 5S genes which we demonstrate in a separate communication to serve as a binding signal for an upstream stimulatory factor.