A comparison of the structural organization of amplified ribosomal DNA from Xenopus mulleri and Xenopus laevis.

Secondary structure maps of long single strands of amplified ribosomal DNA from two closely related species of frogs, Xenopus laevis and X. mulleri, have been compared. The secondary structure pattern of the gene region is identical in both ribosomal DNAs while the patterns in the non-transcribed spacers² differ. In X. mulleri, the spacer shows an extended region without any secondary structure adjacent to the 28 S ribosomal RNA sequence. In contrast, the same region in the X. laevis spacer has extensive secondary structure. A comparison of secondary structure maps and denaturation maps of these two ribosomal DNAs (Brown et al., 1972) reveals that the portion without secondary structure in the X. mulleri spacer corresponds to an early melting A + T-rich region. As in X. laevis ribosomal DNA, Escherichia coli restriction endonuclease (EcoRI) makes two cuts in each repeating unit of amplified ribosomal DNA from X. mulleri. The position of the cleavage sites is identical in the two species as judged from secondary structure mapping of the two classes of EcoRI fragments generated. The small fragments of X. mulleri ribosomal DNA are homogeneous in size with a duplex molecular weight of 3.0 × 10⁶, and contain about 85% of the 28 S ribosomal RNA gene and about 17% of the 18 S ribosomal RNA gene. The large fragments are heterogeneous in size with molecular weights ranging from 4.2 to 4.9 × 10⁶, and contain the remaining portions of the gene regions and the nontranscribed spacer. Heteroduplexes made between large fragments of different lengths show only deletion loops. The position of these loops indicates that the length heterogeneity resides in the non-transcribed spacer region. Electrophoretic analysis of EcoRI digests of chromosomal ribosomal DNA from X. mulleri demonstrates that this DNA is heterogeneous in length as well.     

An electron microscope heteroduplex study of the ribosomal DNAs of Xenopus laevis and Xenopus mulleri

The arrangement of the genes and spacers has been analyzed in ribosomal DNA of Xenopus laevis and Xenopus mulleri by heteroduplex mapping and visualization of ribosomal RNA-DNA hybrids. Heterologous reassoeiated molecules show a characteristic pattern in which two perfectly duplexed regions, whose lengths are those predicted by the known lengths of the 18 S and 28 S genes, are separated by a small substitution loop of about 0.23 × 10⁶ daltons and a large region of partial homology which averages 3.24 × 10⁶ daltons. These mismatched regions are entirely consistent with the known sequence divergence previously described (Brown et al., 1972) for the transcribed and non-transcribed spacer regions of the two rDNAs, respectively. Hybrids of X. laevis rDNA with 18 S and 28 S rRNA contain two duplex regions of the expected lengths for the 18 S and 28 S genes separated by 0.49 × 10⁶ daltons of single-strand DNA. This latter value is the length of the transcribed spacer region between the 18 S and 28 S RNAs that has been measured within the 40 S RNA precursor molecule by secondary structure mapping (Wellauer &; Dawid, personal communication). There is also a longer single-strand region separating one 18 S + 28 S gene set from the next; this is considered to be mainly non-transcribed spacer.We conclude that the 18 S and 28 S genes are separated by about 0.5 × 10⁶ daltons of DNA of which about half is homologous in the two Xenopus species. This region is part of the transcribed spacer. In addition, the longer non-transcribed spacer can be seen to have some homology between the two species; the location of this homology is fairly reproducible between molecules and has been carefully documented by contour length measurements.     

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.     

Secondary structure maps of ribosomal RNA and DNA: I. Processing of Xenopus laevis ribosomal RNA and structure of single-stranded ribosomal DNA

Precursor and mature ribosomal RNA molecules from Xenopus laevis were examined by electron microscopy. A reproducible arrangement of hairpin loops was observed in these molecules. Maps based on this secondary structure were used to determine the arrangement of sequences in precursor RNA molecules and to identify the position of mature rRNAs within the precursors. A processing scheme was derived in which the 40 S rRNA is cleaved to 38 S RNA, which then yields 34 S plus 18 S RNA. The 34 S RNA is processed to 30 S, and finally to 28 S rRNA. The pathway is analogous to that of L-cell rRNA but differs from HeLa rRNA in that no 20 S rRNA intermediate was found. X. laevis 40 S rRNA (M_r = 2.7 × 10⁶) is much smaller than HeLa or L-cell 45 8 rRNA (M_r = 4.7 × 10⁶), but the arrangement of mature rRNA sequences in all precursors is very similar. Experiments with ascites cell 3′-exonuclease show that the 28 S region is located at or close to the 5′-end of the 40 S rRNA.Secondary structure maps were obtained also for single-stranded molecules of ribosomal DNA. The region in the DNA coding for the 40 S rRNA could be identified by its regular structure, which closely resembles that of the RNA. Regions corresponding to the 40 S RNA gene alternate with non-transcribed spacer regions along strands of rDNA. The latter have a large amount of irregular secondary structure and vary in length between different repeating units. A detailed map of the rDNA repeating unit was derived from these experiments.Optical melting studies are presented, showing that rRNAs with a high (G + C) content exhibit significant hypochromicity in the formamide/urea-containing solution that was used for spreading.     

Mitochondrial ribosomal proteins in Xenopus laevis/X. mulleri interspecific hybrids.

The large subunits of mitochondrial ribosomes were isolated from two related frog species, Xenopus laevis and X. mulleri, and their proteins were compared by two-dimensional polyacrylamide gel electrophoresis. Three of the proteins observed in X. laevis are absent from X. mulleri, and four of the proteins observed in X. mulleri are absent from X. laevis. More than these seven such species-specific proteins may occur.Reciprocal crosses between frogs of the two species gave two groups of F1 hybrids. Nuclear genes in these hybrids derive equally from both species, while mitochondrial DNA (and therefore mitochondrial rRNA) derived exclusively from the maternal species. Electrophoretic analyses of the large subunit proteins of these F1 animals revealed that four of the species-specific proteins are present only when their corresponding species was the mother. While this result is consistent with the coding of these four proteins by mitochondrial DNA, it does not provide evidence against nuclear coding of these proteins. A fifth protein is absent from both F1 hybrids. A sixth is present in both F1 hybrids, and a seventh is present only when its corresponding species was the father. We conclude that at least these latter two mitochondrial ribosomal proteins are encoded by nuclear genes.     

The cyclization of Xenopus laevis 5 S DNA

DNA from Xenopus laevis containing the sequences complementary to 5 S RNA has been studied by the formation of folded rings. Maximal cyclization for fragments 1 to 2 μm in length is 45 to 55%. Thus the efficiency of folded ring formation from this tandemly-repeating DNA is about 50%, assuming that all fragments are 5 S DNA. From the ring frequency as a function of the number of nucleotides removed from the 3′ terminals of the shear-broken fragments, one may calculate that the repeating sequence is approximately 750 nucleotides long, a number that agrees with earlier partial denaturation mapping. The circumference of the folded rings confirms this repeating length since most rings correspond to modular size classes of 0.25-μm increments. Fragments 12 μm long cyclize almost as readily as 1 to 2-μm fragments do. Therefore, the length of the regions (g-regions) containing the tandemly-repeating 5 S DNA is more than 12 μm. The folded rings are about as stable to linearization by increasing concentrations of formamide as the duplex DNA is to denaturation. This indicates that the local, non-transcribed, spacer portions which represent the majority (83%) of the nucleotides in the tandemly-repeating unit, are probably homogeneous in sequence. The exonuclease-treated 5 S DNA fragments cyclize more rapidly than phage T7 DNA, and the kinetics are in accord with theoretical expectation.     

Repression of nucleolar organizer activity in an interspecific hybrid of the genus Xenopus

Nucleolar expression in reciprocal hybrids between African frogs of the species Xenopus laevis and X. mulleri has been examined throughout ontogeny. Evidence is presented for the stagespecific regulation of nucleolar expression in these hybrids, and for a maternal effect that operates during the early development. Advantage has been taken of the nucleolar organizer deletion in X. laevis to demonstrate that the uninucleolate conditions of hybrid nuclei at certain developmental stages is the result of an allelic repression of the mulleri nucleolar organizer region by the laevis genome. Differences in nucleolar expression between the reciprocal hybrids and the parental species have been put on a quantitative basis for several selected tissues.     

Chromosomal location of a major tRNA gene cluster of Xenopus laevis

In Xenopus laevis, genes encoding tRNA^Phe, tRNA^Tyr, tRNA ₁ ^Met , tRNA^Asn, tRNA^Ala, tRNA^Leu, and tRNA^Lys are clustered within a 3.18-kb (kilobase) fragment of DNA. This fragment is tandemly repeated some 150 times in the haploid genome and its components are found outside the repeat only to a limited extent. The fragment hybridizes in situ to a single site very near the telomere on the long arm of one of the acrocentric chromosomes of the group comprising chromosomes 13–18. All the chromosomes of this group also hybridize with DNA coding for oocyte-specific 5S RNA. The tRNA gene cluster is slightly proximal to the cluster of 5S RNA genes.We respectfully dedicate this paper to Prof. H. Bauer on the occasion of his 80th birthday.     

Association of Rous sarcoma virus DNA with Xenopus laevis spermatozoa and its transfer to ova through fertilization

Mature Xenopus laevis spermatozoa are capable of binding plasmid pAPrC carrying the complete Rous sarcoma virus (RSV) DNA. Each sperm cell associates, on an average, with 70–160 molecules of the plasmid DNA in a DNase resistant form, if the spermatozoa were exposed to the DNA at a concentration of 1.0–1.4 μg/10⁷ sperm cells. Fertilization with pAPrC-treated spermatozoa induced developmental malformations in 25–30% of embryos. Immunohistochemical analysis of tissue sections from defective animals revealed aberrations in myotomal structures, and increased expression of pp60^src protein in myoblasts, neuronal tube, and epidermis. The presence of characteristic v-src and RSV-long terminal repeat (LTR) sequences in X. laevis DNA was detected by PCR analysis. Embryonic RNA hybridized with a src-specific and an RSV-LTR specific probes indicating expression of the viral DNA. Plasmid DNAs without the v-src gene (pATV9) or completely free of any RSV sequences (pBR322) did not induce any changes in embryonic development. Our results provide evidence that the pBR322-cloned DNA form of the RSV genome associates with frog sperm cells in a DNase-resistant manner suggesting internalization and may be subsequently carried into eggs during the process of artificial fertilization. Correlation between the defective morphogenesis of X. laevis and increased expression of the src gene as well as an interference of RSV DNA with the developmental programs of frog embryos are discussed. © 1996 Wiley-Liss, Inc.     

Recombination of a eukaryotic DNA in bacteria

Single, 824 bp repeating units of Xenopus laevis oocyte-type 5S DNA were inserted into the recombination vectors, λrva and λrvb. When the inserts had the same orientation with respect to the λ chromosomes, Spi^-imm434 recombinants were recovered by selection on a P2, λ double lysogenic host. Because of the structure of the vectors, the crossover point in each recombinant must lie completely within the 5S DNA insert. The physical characteristics of these recombinants were determined by examination of restriction enzyme digests. By use of RecA mutant hosts and the Red^- vector, λrvc, recombination frequencies were measured separately for the bacterial and phage systems.Some of the recombination events resulted in 5S DNA inserts of altered length due to unequal crossovers within repeated sequences in the 5S DNA spacer. The occurrence of just such events in frog 5S DNA had been predicted, based on the structure of 5S DNA and evolutionary considerations.     

The lampbrush chromosomes of Xenopus laevis: preparation,identification, and distribution of 5S DNA sequences

Details are given of a technique for making permanent preparations of the lampbrush chromosomes of Xenopus laevis. Stained preparations allow all 18 bivalent chromosomes to be identified, and a working map showing the major features has been constructed. Fifteen of the Xenopus chromosomes have one telomere conspicuously larger than the other; the two smallest chromosomes, and one other, lack large telomeres. Similar preparations, extracted with RNase and denatured, have been hybridized in situ with a ³H-labelled 5S cRNA probe. Chromosomes can be identified in the resulting autoradiographs. 5S DNA sequences are present at all the larger telomeres and at three of the smaller ones, but are absent from the telomeres at both ends of the two smallest chromosomes. There are also five interstitial sites of hybridization. At one of these, label is on the chromosome axis; at the other four, label extends well away from the axis.     

Distribution of 18+28S ribosomal genes in mammalian genomes

In situ hybridization with ³H 18S and 28S ribosomal RNA from Xenopus laevis has been used to study the distribution of DNA sequences coding for these RNAs (the nucleolus organizing regions) in the genomes of six mammals. Several patterns of distribution have been found: 1) A single major site (rat kangaroo, Seba's fruit bat), 2) Two major sites (Indian muntjac), 3) Multiple sites in centromeric heterochromatin (field vole), 4) Multiple sites in heterochromatic short arms (Peromyscus eremicus), 5) Multiple sites in telomeric regions (Chinese hamster). — The chromosomal sites which bind ³H 18S and 28S ribosomal RNA correspond closely to the sites of secondary constrictions where these are known. However, the correlation is not absolute. Some secondary constrictions do not appear to bind ³H ribosomal RNA. Some regions which bind ribosomal RNA do not appear as secondary constrictions in metaphase chromosomes. — Although the nucleolus organizing regions of most mammalian karyotypes are found on the autosomes, the X chromosomes in Carollia perspicillata and C. castanea carry large clusters of sequences complementary to ribosomal RNA. In situ hybridization shows that the Y chromosome in C. castanea also has a large nucleolus organizing region.     

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.     

Cloning,sequencing and expression of the two genes encoding the mitochondrial single-stranded DNA-binding protein in Xenopus laevis

In Xenopus laevis the single-stranded DNA binding protein imported into the mitochondria consists of two highly related polypeptides. The establishment of the genomic nucleotide sequences reveals that they are encoded by two different genes, XLSSB1 and XLSSB2. The deduced amino acid sequence is identical to the direct amino acid sequence determined by Edman degradation of the mitochondrial polypeptides [Ghrir, R., Lecaer, J.P., Dufresne, C. and Gueride, M. (1991) Primary structure of the two variants of Xenopus laevis mtSSB, a mitochondrial DNA binding protein. Arch. Biochem. Biophys. 291, 395–400]. Both genes are organized in seven exons and six introns, the sequence of the peptide leader is interrupted by an intervening sequence (intron 2). The exon/intron junctions are in exactly conserved positions, splitting the same codon. A high level of identity is observed between corresponding introns of the two genes over part or most of their lengths. Structural features of intronic sequences reveal multiple rearrangements and exchanges during the evolution of X. laevis species. A CCAAT box and the potential regulatory elements NRF-2 and Sp 1 are observed in the 5′-flanking region of both genes. During oogenesis, XLSSB gene expression is correlated with the replicative activity of the mitochondrial DNA.