Chromosomal mapping of Xenopus 5S genes: somatic-type versus oocyte-type.

Xenopus 5S RNA genes exhibit a pattern of differential expression during development in which some members (oocyte-type) are transcribed only in oocytes, while others (somatic-type) are expressed in both oocytes and somatic cells. Using cloned DNA probes specific for each gene type, we determined the positions of these genes on Xenopus metaphase chromosomes by in situ hybridization. Somatic-type 5S genes in both X. laevis and X. borealis are located at the distal end of the long arm of only one chromosome (number 9). The oocyte-type 5S RNA genes are found at the distal ends of the long arms of most Xenopus chromosomes, including chromosome 9. Thus, large scale differences in chromosomal location cannot explain the selective expression of these genes, as suggested previously.     

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.     

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.     

Reiterated transfer RNA genes of Xenopus laevis

The proportion of the Xenopus laevis genome complementary to “7 S” RNA, unfractionated transfer RNA and some selected aminoacyl-tRNAs, and the sequence complexity of these RNA species, have been determined by following the kinetics of RNA-DNA hybridization on filters under conditions of RNA excess at optimum rate temperature. For hybridization of aminoacyl-labelled tRNAs, conditions for optimum aminoacylation were first determined and, where necessary, aminoacyl-tRNAs were treated with nitrous acid to prevent discharge during annealing. Neither the extent nor rate of hybridization was affected by this treatment.“7 S” RNA, coded for by 580 genes per haploid complement of chromosomes, reacts like a single family of nucleotide sequences, whereas about 43 basic tRNA sequences are coded for by at least 7800 genes. If hybrids are not treated with RNase A, the apparent tDNA redundancy is some 23% greater but no more nucleotide sequences are detectable. Taken together, the results suggest that each tRNA sequence is, on average, 200-fold reiterated.The reiteration varies, however, for different aminoacyl-tRNAs. Thus, hybridization resolves only one valyl-tRNA which is coded for by 240 genes, but at least four leucyl-tRNA sequences can be distinguished by hybridization, each of which is on average 90-fold reiterated. Reiteration also varies for the two methionyl-tRNAs detectable both by hybridization and by reversed phase chromatography: tRNA₁^Met and tRNA₂^Met are 310- and 170-fold reiterated, respectively, and each is kinetically homogeneous. These saturation values are almost exactly additive and are not influenced by the presence of other tRNA species. Thus the results suggest that Xenopus tRNAs are no more heterogeneous than would be predicted by the genetic code, despite the high but variable multiplicity of tRNA cistrons.     

High resolution mapping of Xenopus laevis 5S and ribosomal RNA genes by EM in situ hybridization

We have developed a modification of in situ hybridization at the electron microscope level that permits simultaneous detection of at least two sequences. Probes are labelled with either biotin or AAF and detected with two distinct sizes of colloidal gold. This protocol has been applied to map the positions of Xenopus laevis oocyte-type 5S genes relative to ribosomal precursor genes in several independently derived cell lines. The results for the line TRXO, which expresses some oocyte 5S RNA, indicate that this inappropriate expression is not due to translocation from telomeric sites into the nucleolus organizer, as previously hypothesized. In addition we found that four other Xenopus cell lines, none of which express these genes, also contain distinct 5S oocyte translocations. These results suggest that an alteration in chromosome position is insufficient to result in gene activation and that sequences which are telomeric-proximal are exceptionally prone to translocation.     

Organization of 5S genes in chromatin of Xenopus laevis.

The chromatin organization of the genes coding for 5S RNA in Xenopus laevis has been investigated with restriction endonucleases and micrococcal nuclease. Digestion of nuclei from liver, kidney, blood and kidney cells maintained in culture with micrococcal nuclease reveals that these Xenopus cells and tissues have shorter nucleosome repeat lengths than the corresponding cells and tissues from other higher organisms. 5S genes are organized in nucleosomes with repeat lengths similar to those of the bulk chromatin in liver (178 bp) and cultured cells (165 bp); however, 5S gene chromatin in blood cells has a shorter nucleosome repeat (176 bp) than the bulk of the genome in these cells (184 bp). From an analysis of the 5S DNA fragments produced by extensive restriction endonuclease cleavage of chromatin in situ, no special arrangement of the nucleosomes with respect to the sequence of 5S DNA can be detected. The relative abundance of 5S gene multimers follows a Kuhn distribution, with about 57% of all HindIII sites cleaved. This suggests that HindIII sites can be cleaved both in the nucleosome core and linker regions.     

Decay of the oocyte-type heat shock response of Xenopus laevis

Xenopus oocytes have a complex heat shock response. During transition of the oocyte into fertilized egg, the heat shock response undergoes several qualitative and quantitative changes culminating in its complete extinction. Heat shock induces oocytes to synthesize four heat shock proteins (hsps): 83, 76, 70, and 57. After ovulation, two additional proteins (hsps 22 and 16) are inducible. The heat shock response of spawned eggs can be modified by changing the ionic configuration of the external medium and by adding pyruvate and oxaloacetate to the media. Since Xenopus eggs do not synthesize mRNA, these modifications to the external medium apparently alter the utilization of preexisting messenger RNAs in protein synthesis. Artificial activation terminates inducibility of hsps 76, 57, and 16 and diminishes the hsp 70 response. Two new heat shock proteins-66 and 48-are also inducible in artificially activated eggs. Fertilization, on the other hand, terminates the heat shock response; no hsps can be induced. However, hsp 70 appears to be made constitutively in fertilized eggs. RNA blot analyses reveal that oogenic hsp 70 messenger RNA is retained in eggs and early embryos. This messenger is apparently used for heat-induced synthesis of hsp 70 before fertilization and for constitutive synthesis of hsp 70 in zygotes.