Very long-lived messenger RNA in ovaries of Xenopus laevis

It is possible to label with radioactivity newly synthesized ovarian RNA after intraperitoneal injection of [³H]guanosine and [³H]uridine into immature Xenopus laevis, if ovaries in which only previtellogenic stage 1 oocytes are present. Following the amount of radioactivity in the ovarian pool of acid-soluble precursors indicates a complete clearance of acid-soluble radioactivity within 15–20 days after injection. Incorporation of radioactivity into total RNA (which is almost exclusively 4 and 5S RNAs at this stage) and poly(A)⁺ RNA ceases between 15 and 20 days after injection, but the total amount of radioactivity in these RNA fractions does not decline appreciably over the next 18 months. During this time, the ovary grows and develops since stage 6 oocytes eventually appear and there is a 10- to 20-fold increase in total RNA content, which changes in composition from almost exclusively (95%) 4 and 5S RNAs to mainly (75%) 18 and 28S RNAs. Thus, despite continued growth and development, radioactive RNA molecules synthesized during previtellogenesis survive for lengths of time commensurate with the length of oogenesis (1–2 years). Although very limited (<7%) reincorporation of radioactivity into RNA is detected, it cannot alone account for the stability of the label in poly(A)⁺ RNA. These results are interpreted as indicative of synthesis during previtellogenesis of tRNA, 5SrRNA, and messenger RNA molecules which are very long-lived.     

Location of the genes for 5S ribosomal RNA in Xenopus laevis

In situ hybridization of 5S RNA and cRNA transcribed in vitro from Xenopus laevis 5S DNA shows that 5S DNA is localized at or near the telomere region of the long arm of many, if not all, of the X. laevis chromosomes. No 5S DNA is detected near the nucleolus organizer in the normal X. laevis chromosome complement, but in a X. laevis kidney cell line, 5S DNA is found at the distal end of the secondary constriction. The arrangement of 5S DNA in several types of interphase nuclei is described. — During the pairing stages of meiosis the telomeres of most or perhaps all of the chromosomes become closely associated so that the regions containing 5S DNA form a single cluster. This close association might be either a cause or a result of the presence of the similar sequences of 5S DNA on many telomeres. It suggests that the uniformity of 5S sequences on non-homologous chromosomes might be maintained by crossing-over between the chromosomes.     

Persistence of nonribosome bound 5 S RNA in full-grown oocytes of Xenopus laevis

The 4 and 5 S RNA containing 42 S ribonucleoprotein (RNP) particles characteristic of previtellogenic and white oocytes cannot be detected in full-grown oocytes. When full-grown oocyte RNPs are separated on sucrose gradients 4 and 5 S RNA cannot be detected in the 42 S region. However, not all of the 5 S RNA stored during early oogenesis is incorporated into ribosomes at later stages. A substantial pool (20% of the total) of 5 S RNA remains in a non-ribosome-bound fraction sedimenting at about 7 S in full-grown oocytes.     

The genomic organization of dispersed tRNA and 5 S RNA genes in Xenopus laevis

The 5 S DNAs and several tDNAs of Xenopus laevis reside primarily in large clusters of tandem repeating units. We have discovered that a substantial number of these genes, along with portions of their adjacent spacer sequences, are also located in dispersed genomic locations apart from the major clusters. This was accomplished by "null-digesting" total genomic DNA with restriction enzymes that do not cut within the X. laevis tDNA or 5 S DNA major repeats. The tDNA and 5 S DNA main clusters therefore remain intact and can be easily separated on gels from the dispersed tDNAs and 5 S DNAs present as low molecular weight restriction fragments. Probing these smaller fragments with different portions of the major repeats has revealed that many of the dispersed genes are organized differently from the corresponding tDNAs and 5 S DNAs of the primary clusters. Some of the fragments containing dispersed genes are actually present in multiple copies. In addition, many tDNA null-digestion fragments contain more than one type of tRNA coding region. One set of "dispersed" tDNAs actually comprises a tandemly arranged minor tDNA family which has retained the same repeat length (3.18 kb) as the major tDNA family, but has a substantially different organization. There is significant population polymorphism in the organization of the dispersed tDNAs and 5 S DNAs. Dispersed genes that appear to be derived from clusters of tandem repeats ("orphons") have been described for several gene families in invertebrates. The occurrence of this phenomenon in vertebrates as well, suggests that such dispersed genes may be a general feature of all eukaryotic genomes.