Oocyte-type 5S Research Articles

The characterization of the TFIIIA synthesized in somatic cells of Xenopus laevis.

In somatic cells of Xenopus, transcription of the TFIIIA gene initiates greater than 200 bp upstream from the start site used in oocytes. The resultant mRNA encodes a protein, S-TFIIIA, that is 22 amino acids longer at its amino terminus than the abundant form of TFIIIA in oocytes (O-TFIIIA). S-TFIIIA binds the 5S RNA gene and 5S RNA, and both O- and S-TFIIIA promote the formation of stable transcription complexes on oocyte-type 5S RNA genes in an oocyte nuclear extract. We have not found any functional difference between the two forms of TFIIIA. Different transcription start sites suggest differential promoter usage--one in oocytes that permits high levels of gene activity and another that is used in somatic cells for low-level TFIIIA mRNA synthesis.

Chromosomal footprinting of transcriptionally active and inactive oocyte-type 5S RNA genes ofXenopus laevis

The chromatin structure of the Xenopus oocyte-specific 5S rRNA genes was examined at high resolution in immature oocyte and somatic cell chromosomes by DNase I footprinting. On oocyte chromatin, where the genes are active, the cleavage preferences over the entire gene region showed a periodic pattern of sensitivity and were dramatically different from the patterns obtained with deproteinized DNA or somatic cell chromatin. Further, the normal binding site for TFIIIA over the internal promoter region was preferentially sensitive to cleavage, indicating that TFIIIA was not bound in the manner predicted by in vitro experiments. In somatic cell chromatin, the oocyte-type 5S genes displayed a cleavage pattern largely similar to deproteinized DNA suggesting the absence of positioned nucleosomes on these inactive genes, although the presence of uncharacterized repressor complexes could not be ruled out. These data are discussed in terms of potential forms of the chromatin structure and alternative mechanisms of oocyte-type gene activation.

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.

Two TFIIIA activities regulate expression of the Xenopus 5S RNA gene families.

Immunoblotting experiments with polyclonal and monoclonal anti-transcription factor IIIA (TFIIIA) antibodies reveal different electrophoretic forms of TFIIIA in extracts from immature and mature oocytes of Xenopus laevis. The well-characterized 39-kD TFIIIA species is present in approximately 10(12) copies per cell in stage I-III previtellogenic oocytes and declines in abundance by 10- to 20-fold during oogenesis. An immunologically related protein of apparent molecular mass of 42 kD is present at 2-4% of the level of 39-kD TFIIIA in immature oocytes, and the level of this protein increases dramatically during oogenesis. Both the 39- and 42-kD proteins are complexed with 5S RNA in 7S ribonucleoprotein (RNP) particles. High-level transcription of the oocyte-type 5S genes in vitro requires 39-kD immature oocyte TFIIIA, whereas both 39-kD TFIIIA and the mature oocyte TFIIIA species of 42 kD support somatic-type 5S transcription. TFIIIA of 42 kD does not support oocyte-type 5S transcription in a fractionated transcription system derived from mature oocytes. Both proteins, however, bind the oocyte-type and somatic-type genes with comparable affinities and exhibit similar DNase footprints on both genes. These results suggest a model for the developmental regulation of 5S RNA gene transcription where 42-kD TFIIIA serves as an activator of somatic-type 5S transcription and as a repressor of oocyte-type transcription during early embryogenesis.

Biochemical research on oogenesis. RNA accumulation in the oocytes of the newt Pleurodeles waltl

We have compared the accumulation of 5S RNA and tRNA in oocytes of Pleurodeles waltl with the corresponding process previously studied in Xenopus laevis. 5S RNA synthesis is regulated similarly in both species since different families of 5S RNA genes are transcribed in oocytes and in somatic cells of P. waltl, as in those of X. laevis. Previtellogenic oocytes of P. waltl contain only one prominent kind of storage particles (thesaurisomes). In contrast, X. laevis oocytes of the same size contain two major classes of thesaurisomes, sedimenting at 42S and 7S. The more abundant particles found in P. waltl oocytes are homologous to the larger thesaurisomes (42S) of X. laevis, but they have a lower sedimentation coefficient and a higher tRNA/5S RNA molar ratio than their X. laevis counterparts. Small amounts of particles which we think to be homologous to the 7S particles of X. laevis are present in previtellogenic oocytes of P. waltl. Therefore, the storage function of the 7S particle protein (TFIIIA) is only marginal in this species. In X. laevis oocytes TFIIIA has a second function. It acts as a positive transcription factor involved in the developmentally regulated expression of the 5S RNA genes. In X. laevis expression of the oocyte-type 5S RNA genes is accompanied by a massive accumulation of TFIIIA. This is not the case in P. waltl.

Transcriptionally inactive oocyte-type 5S RNA genes of Xenopus laevis are complexed with TFIIIA in vitro.

An extract from whole oocytes of Xenopus laevis was shown to transcribe somatic-type 5S RNA genes approximately 100-fold more efficiently than oocyte-type 5S RNA genes. This preference was at least 10-fold greater than the preference seen upon microinjection of 5S RNA genes into oocyte nuclei or upon in vitro transcription in an oocyte nuclear extract. The approximately 100-fold transcriptional bias in favor of the somatic-type 5S RNA genes observed in vitro in the whole oocyte extract was similar to the transcriptional bias observed in developing Xenopus embryos. We also showed that in the whole oocyte extract, a promoter-binding protein required for 5S RNA gene transcription, TFIIIA, was bound both to the actively transcribed somatic-type 5S RNA gene and to the largely inactive oocyte-type 5S RNA genes. These findings suggest that the mechanism for the differential expression of 5S RNA genes during Xenopus development does not involve differential binding of TFIIIA to 5S RNA genes.

Transcriptionally inactive oocyte-type 5S RNA genes of Xenopus laevis are complexed with TFIIIA in vitro.

An extract from whole oocytes of Xenopus laevis was shown to transcribe somatic-type 5S RNA genes approximately 100-fold more efficiently than oocyte-type 5S RNA genes. This preference was at least 10-fold greater than the preference seen upon microinjection of 5S RNA genes into oocyte nuclei or upon in vitro transcription in an oocyte nuclear extract. The approximately 100-fold transcriptional bias in favor of the somatic-type 5S RNA genes observed in vitro in the whole oocyte extract was similar to the transcriptional bias observed in developing Xenopus embryos. We also showed that in the whole oocyte extract, a promoter-binding protein required for 5S RNA gene transcription, TFIIIA, was bound both to the actively transcribed somatic-type 5S RNA gene and to the largely inactive oocyte-type 5S RNA genes. These findings suggest that the mechanism for the differential expression of 5S RNA genes during Xenopus development does not involve differential binding of TFIIIA to 5S RNA genes.

The positive transcription factor of the 5S RNA gene proteolyses during direct exchange between 5S DNA sites.

We have examined the association, dissociation, and exchange of the 5S specific transcription factor (TFIIIA) with somatic- and oocyte-type 5S DNA. The factor associates faster with somatic than with oocyte 5S DNA, and the rate of complex formation is accelerated by vector DNA. Once formed, the TFIIIA-5S DNA complex is stable for greater than 4 h in the absence of free 5S DNA, and its dissociation is identical for somatic and for oocyte 5S DNA. In the presence of free 5S DNA, the factor transfers promptly from the complex to the free 5S DNA site. Unexpectedly, the direct exchange of factor between 5S DNA sites leads to proteolysis at the C-terminal arm of TFIIIA.

Early replication and expression of oocyte-type 5S RNA genes in a Xenopus somatic cell line carrying a translocation.

In Xenopus somatic cells, the somatic-type 5S RNA genes replicate early in S phase, bind the transcription factor TFIIIA, and are expressed; in contrast, the late replicating oocyte-type genes do not bind TFIIIA and are transcriptionally inactive. These facts support a model in which the order of replication of the somatic-type versus the oocyte-type 5S genes causes their differential expression in somatic cells due to sequestration of TFIIIA by the early-replicating somatic genes. Here we provide further evidence for the model by showing that in one Xenopus cell line in which some oocyte-type 5S genes are translocated, some oocyte-type 5S genes replicate early and are expressed.

Differential order of replication of Xenopus laevis 5S RNA genes.

In Xenopus laevis there are two multigene families of 5S RNA genes: the oocyte-type 5S RNA genes which are expressed only in oocytes and the somatic-type 5S RNA genes which are expressed throughout development. The Xenopus 5S RNA replication-expression model of Gottesfeld and Bloomer (Cell 28:781-791, 1982) and Wormington et al. (Cold Spring Harbor Symp. Quant. Biol. 47:879-884, 1983) predicts that the somatic-type 5S RNA genes replicate earlier in the cell cycle than do the oocyte-type genes. Hence, the somatic-type 5S RNA genes have a competitive advantage in binding the transcription factor TFIIIA in somatic cells and are thereby expressed to the exclusion of the oocyte-type genes. To test the replication-expression model, we determined the order of replication of the oocyte- and somatic-type 5S RNA genes. Xenopus cells were labeled with bromodeoxyuridine, stained for DNA content, and then sorted into fractions of S phase by using a fluorescence-activated cell sorter. The newly replicated DNA containing bromodeoxyuridine was separated from the lighter, unreplicated DNA by equilibrium centrifugation and was hybridized with DNA probes specific for the oocyte- and somatic-type 5S RNA genes. In this way we found that the somatic-type 5S RNA genes replicate early in S phase, whereas the oocyte-type 5S RNA genes replicate late in S phase, demonstrating a key aspect of the replication-expression model.

Differential Order of Replication of Xenopus laevis 5S RNA Genes

In Xenopus laevis there are two multigene families of 5S RNA genes: the oocyte-type 5S RNA genes which are expressed only in oocytes and the somatic-type 5S RNA genes which are expressed throughout development. The Xenopus 5S RNA replication-expression model of Gottesfeld and Bloomer (Cell 28:781-791, 1982) and Wormington et al. (Cold Spring Harbor Symp. Quant. Biol. 47:879-884, 1983) predicts that the somatic-type 5S RNA genes replicate earlier in the cell cycle than do the oocyte-type genes. Hence, the somatic-type 5S RNA genes have a competitive advantage in binding the transcription factor TFIIIA in somatic cells and are thereby expressed to the exclusion of the oocyte-type genes. To test the replication-expression model, we determined the order of replication of the oocyte- and somatic-type 5S RNA genes. Xenopus cells were labeled with bromodeoxyuridine, stained for DNA content, and then sorted into fractions of S phase by using a fluorescence-activated cell sorter. The newly replicated DNA containing bromodeoxyuridine was separated from the lighter, unreplicated DNA by equilibrium centrifugation and was hybridized with DNA probes specific for the oocyte- and somatic-type 5S RNA genes. In this way we found that the somatic-type 5S RNA genes replicate early in S phase, whereas the oocyte-type 5S RNA genes replicate late in S phase, demonstrating a key aspect of the replication-expression model.

Temporal order of replication of Xenopus laevis 5S ribosomal RNA genes in somatic cells.

The timing of replication of both the somatic and oocyte-type 5S ribosomal RNA genes of Xenopus laevis was determined in cultured cells by using 5-bromodeoxyuridine labeling of DNA coupled with a retroactive synchrony technique employing the fluorescence-activated cell sorter (FACS). The somatic genes replicate very early in S phase, while the oocyte genes replicate very late. These experiments provide direct evidence for a model [Gottesfeld, J. & Bloomer, L. S. (1982) Cell 28, 781-791] in which the transcription-activating factor TFIIIA is required at the time of Xenopus 5S rRNA gene replication to facilitate transcription of these genes.

Torsional stress induces an S1 nuclease-hypersensitive site within the promoter of the Xenopus laevis oocyte-type 5S RNA gene.

The internal promoter of the Xenopus laevis oocyte-type 5S RNA gene is preferentially cleaved by S1 and Bal-31 nucleases in plasmid DNA. S1 nuclease sensitivity is largely dependent on supercoiling; however, Bal-31 cleaves within the 5S RNA gene in linear as well as in supercoiled DNA. The S1 nuclease-hypersensitive site is centered at position +48-52 of the gene at the 5' boundary of the promoter. A DNAase I-hypersensitive site is induced at this position upon binding of the transcription factor, TFIIIA, specific for the 5S RNA gene. The somatic-type 5S RNA gene promoter is not preferentially cleaved by S1 nuclease or Bal-31 nuclease in supercoiled DNA, nor does TFIIIA induce a DNase I site at position +50. This differential promoter response may be related to a 4-fold difference in TFIIIA affinity between the oocyte and somatic 5S RNA genes.

Cytoplasmic regulation of 5S RNA genes in nuclear-transplant embryos.

In the normal development of Xenopus laevis, genes for oocyte-type and somatic-type 5S RNAs are both expressed in late blastulae. Estimates of rates of synthesis indicate that the oocyte-type genes (5Sooc) undergo at least a 100-fold reduction in transcriptional activity between the end of oogenesis and the late blastula stage, and at least a further 20-fold reduction during gastrulation. When neurula nuclei, with inactive 5Sooc genes, were transplanted to enucleated eggs, the resulting nuclear-transplant embryos showed a reactivation of 5Sooc genes in blastulae and a subsequent inactivation after this stage. This effect is not explicable by a differential stability of the two types of 5S RNA. We conclude that egg or early embryo cytoplasm must contain components which can continuously regulate 5S gene expression, and that the mechanism by which 5Sooc genes are developmentally inactivated does not persist through mitosis in early embryos. These results have been obtained by a new procedure in which oocyte- and somatic-type 5S RNAs are separated in a 4 M urea-15% acrylamide gel.

Regular arrangement of nucleosomes on 5S rRNA genes in Xenopus laevis.

The chromatin structure of the oocyte-type 5S RNA genes in Xenopus laevis was investigated. Blot hybridization analysis of DNA from micrococcal nuclease digests of erythrocyte nuclei showed that 5S DNA has the same average nucleosome repeat length, 192 +/- 4 base pairs, as two Xenopus satellite DNAs and bulk erythrocyte chromatin. The positions of nuclease-sensitive regions in the 5S DNA repeats of purified DNA and chromatin from erythrocytes were mapped by using an indirect end-labeling technique. Although most of the sites cleaved in purified DNA were also cleaved in chromatin, the patterns of intensities were strikingly different in the two cases. In 5S chromatin, three nuclease-sensitive regions were spaced approximately a nucleosome length apart, suggesting a single, regular arrangement of nucleosomes on most of the 5S DNA repeats. The observed nucleosome locations are discussed with respect to nucleotide sequences known to be important for expression of 5S RNA. Because the preferred locations appear to be reestablished in each repeating unit, despite spacer length heterogeneity, we suggest that the regular chromatin structure reflects the presence of a sequence-specific DNA-binding component on inactive 5S RNA genes.

Regular arrangement of nucleosomes on 5S rRNA genes in Xenopus laevis.

The chromatin structure of the oocyte-type 5S RNA genes in Xenopus laevis was investigated. Blot hybridization analysis of DNA from micrococcal nuclease digests of erythrocyte nuclei showed that 5S DNA has the same average nucleosome repeat length, 192 +/- 4 base pairs, as two Xenopus satellite DNAs and bulk erythrocyte chromatin. The positions of nuclease-sensitive regions in the 5S DNA repeats of purified DNA and chromatin from erythrocytes were mapped by using an indirect end-labeling technique. Although most of the sites cleaved in purified DNA were also cleaved in chromatin, the patterns of intensities were strikingly different in the two cases. In 5S chromatin, three nuclease-sensitive regions were spaced approximately a nucleosome length apart, suggesting a single, regular arrangement of nucleosomes on most of the 5S DNA repeats. The observed nucleosome locations are discussed with respect to nucleotide sequences known to be important for expression of 5S RNA. Because the preferred locations appear to be reestablished in each repeating unit, despite spacer length heterogeneity, we suggest that the regular chromatin structure reflects the presence of a sequence-specific DNA-binding component on inactive 5S RNA genes.

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.

Control of 5S RNA transcription in Xenopus somatic cell chromatin: activation with an oocyte extract.

A chromatin fraction enriched for Xenopus 5S RNA genes has been isolated by restriction endonuclease digestion and sucrose gradient velocity sedimentation. Soluble chromatin sedimenting at 70-80S contains approximately 50% of the oocyte-expressed 5S RNA genes and only 1.5-3% of total chromatin DNA; this represents a 15- to 30-fold purification of the 5S genes. Such chromatin isolated from somatic cells (blood and cultured kidney cells) retains the transcriptionally-inactive state of the oocyte-expressed 5S genes. Soluble chromatin from somatic cells prepared by micrococcal nuclease digestion also retains the inactive state of the oocyte-type 5S genes. It is likely that the level of chromatin structure responsible for inactivity of the oocyte genes in somatic cells is the nucleosome or short chains of nucleosomes and not supranucleosomal structures. The oocyte-type genes can be rendered transcriptionally active in somatic cell chromatin either by salt extraction of some chromosomal proteins or by treatment with the ion exchange resin Dowex A50W-X2. Alternatively, activation of these genes can be achieved by incubating somatic cell chromatin or nuclei with an extract prepared from Xenopus oocytes. This effect is not specific for 5S RNA genes as the transcription of other small RNAs (including pre-tRNA) is stimulated by the oocyte extract. The activating factor(s) is resistant to micrococcal nuclease, nondialyzable, heat labile and sensitive to trypsin; thus it is highly likely to be a protein or a group of proteins. Partial purification of the activating factor(s) has been achieved by ion exchange chromatography.

Oocyte extracts reactivate developmentally inert Xenopus 5S genes in somatic nuclei.

In Xenopus and other amphibians, there are two major classes of 5S RNA gene1. One class, which codes for somatic-type 5S RNA, is expressed in both oocytes and somatic cells2; the other, which is 50 times more abundant3 and which codes for oocyte-type 5S RNA, is expressed in oocytes but not in somatic cells2,4. The developmentally inactivated oocyte-type genes are reactivated when erythrocyte nuclei are injected into most oocytes (activating oocytes)5. As the injected nuclei are induced to take on the natural activity of the host cell, the activation of oocyte-type 5S genes may represent a normal control mechanism. A special opportunity to investigate this mechanism is provided by the existence of certain individual animals whose oocytes consistently fail to activate 5S genes (non-activating oocytes). We have injected extracts of activating or non-activating oocytes, together with erythrocyte nuclei, into oocytes of each kind. We show here that the transcription of 5S genes is induced by extracts of activating oocytes (positive regulation) and is not inhibited by similar extracts of non-activating oocytes. The activating factor is sensitive to protease and heat treatment, suggesting that a protein is involved.

Developmental inactivity of 5S RNA genes persists when chromosomes are cut between genes.

Xenopus and other amphibians possess two major classes of 5S RNA genes1. One class, which codes for somatic-type 5S RNA, is expressed in both oocytes and somatic cells2. The other class, which is 50 times more abundant and encodes oocyte-type 5S RNA, is expressed in oocytes but not in somatic cells2–4. The oocyte-type 5S genes are located in tandem arrays of hundreds of genes at the ends of most chromosomes5, and it has been postulated that the lack of expression of oocyte-type 5S RNA genes in somatic cells is due to some general characteristic of the chromosomal region which the genes occupy6, for example, a folding into heterochromatin or higher-order structures. An alternative possibility is that the inactivity of these genes is controlled individually, and is independent of adjacent genetic material. We have now distinguished between these two possibilities by testing the transcription of oocyte-type 5S RNA genes either in their normal arrangement on intact chromosomes or after each gene has been separated from its neighbours by micrococcal nuclease digestion. Transcription was assayed by injecting intact nuclei, containing the 5S RNA genes as chromatin, into Xenopus oocytes7. We find that the developmental inactivity of oocyte-type 5S RNA genes is retained after the chromatin is cleaved into fragments the size of individual gene repeats.