Long-range organization and sequence-directed curvature of Xenopus laevis satellite 1 DNA.

We have investigated the long-range organization and the intrinsic curvature of satellite 1 DNA, an unusual tandemly-repeated DNA family of Xenopus laevis presenting sequence homologies to SINEs. PFGE was used in combination with frequent-cutter restriction enzymes not likely to cut within satellite 1 DNA and revealed that almost all the repeating units are tandemly organized to form large arrays (200 kb to 2 Mb) that are marked by restriction length polymorphism and contain intra-array domains of sequence variation. Besides that, we have analysed the secondary structure of satellite 1 DNA by computer modelling. Theoretical maps of curvature obtained from three independent models of DNA bending (the dinucleotide wedge model of Trifonov, the junction model of Crothers and the model of de Santis) showed that satellite 1 DNA is intrinsically curved and these results were confirmed experimentally by polyacrylamide gel electrophoresis. Moreover, we observed that this bending element is highly conserved among all the members of the satellite 1 DNA family that are accessible to analysis. A potential genetic role for satellite 1 DNA based on this unusual structural feature is discussed.     

Tandemly repeated pentanucleotides in DNA sequences of eucaryotes.

Genetic sequence data banks were scanned in order to retrieve tandemly repeated pentanucleotides (pnts). It was found that among 102 (=(1024-4)/2/5) possible distinct pnts roughly each fourth is involved in tandem repeats. It is shown that tandemly repeated pnts are composed of frequently occurring di- and trinucleotides and that those pnts which occur frequently in the form of mono- or di-pnts form also tandem repeats either in the form of satellites or in the form of shorter tandem repeats. Human satellite III is taken as a specific example. It is shown that the first guanine within GG-AAT pnt exhibits the highest mutability. Sequential distribution of base changes gives evidence that the mutations do not occur at random positions but in a correlated fashion so that long stretches of original pnts remain intact. It is found that pnts related to the satellite III are present in introns and flanking regions of some structural genes, but are not preserved between orthologous genes of related species. The results corroborate the most plausible mechanism of their evolution--rapid amplification followed by successive divergence of repeat units by various mutational processes.     

DNA ligase I from Xenopus laevis eggs.

We have purified the major DNA ligase from Xenopus laevis eggs and raised antibodies against it. Estimates from SDS PAGE indicate that this DNA ligase is a 180 kDa protein. This enzyme is similar to the mammalian type I DNA ligase which is presumed to be involved in DNA replication. We have also analysed DNA ligase activity during X. laevis early development. Unfertilized eggs contain the highest level of activity reflecting the requirement for a large amount of DNA replicative enzymes for the period of intense replication following fertilization. In contrast with previous studies on the amphibians axolotl and Pleurodeles, the major DNA ligase activity detected during X. laevis early development is catalysed by a single enzyme: DNA ligase I. And the presence of this DNA ligase I in Xenopus egg before fertilization clearly demonstrates that the exclusion process of two forms of DNA ligase does not occur during X. laevis early development.     

Simple repeated sequences in human satellite DNA.

In an extensive analysis, using a range of restriction endonucleases, HinfI and TaqI were found to differentiate satellites I, II and III & IV. Satellite I is resistant to digestion by TaqI, but is cleaved by HinfI to yield three major fragments of approximate size 770, 850 and 950bp, associated in a single length of DNA. The 770bp fragment contains recognition sites for a number of other enzymes, whereas the 850 and 950bp fragments are "silent" by restriction enzyme analysis. Satellite II is digested by HinfI into a large number of very small (10-80bp) fragments, many of which also contain TaqI sites. A proportion of the HinfI sites in satellite II have the sequence 5'GA(GC)TC. The HinfI digestion products of satellites III and IV form a complete ladder, stretching from 15bp or less to more than 250bp, with adjacent multimers separated by an increment of 5bp. The ladder fragments do not contain TaqI sites and all HinfI sites have the sequence 5'GA(AT)TC. Three fragments from the HinfI ladder of satellite III have been sequenced, and all consist of a tandemly repeated 5bp sequence, 5'TTCCA, with a non-repeated, G+C rich sequence, 9bp in length, at the 3' end.     

Isolated clusters of paired tandemly repeated sequences in the Xenopus laevis genome.

There exist in the Xenopus laevis genome clusters of tandemly repeated DNA sequences, consisting of two types of 393-base-pair repeating unit. Each such cluster contains several units of one of these paired tandem repeats (PTR-1), followed by several units of the other repeat (PTR-2). The number of repeats of each type is variable from cluster to cluster and averages about seven of each type per cluster. Every cluster has ca. 1,000 base pairs of common left flanking sequence (adjacent to the PTR-1 repeats) and 1,000 base pairs of common right flanking sequence (adjacent to the PTR-2 repeats). Beyond these common flanks, the DNA sequences are different in the eight cloned genomic fragments we have studied. Thus, the hundreds of PTR clusters in the genome are dispersed at apparently unrelated sites. Nucleotide sequences of representative PTR-1 and PTR-2 repeats are 64% homologous. These sequences do not reveal an obvious function. However, the related species X. mulleri and X. borealis have sequences homologous to PTR-1 and PTR-2, which show the same repeat lengths and genomic organization. This evolutionary conservation suggests positive selection for the clusters. Maintenance of these sequences at dispersed sites imposes constraints on possible mechanisms of concerted evolution.     

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.     

Characterisation of bacterial clones containing DNA sequences derived from Xenopus laevis vitellogenin mRNA.

A 1700 nucleotide DNA sequence derived from Xenopus vitellogenin mRNA has been cloned in the bacterial plasmid pBR322. The identity of the cloned sequence was verified in two ways. Firstly, the plasmid DNA was shown to hybridise to an RNA of the correct size (6,700 nucleotides). This was shown by in situ hybridisation to electrophoretically separated RNA and also by the formation of "R-loops" with purified vitellogenin mRNA. Then, using a novel procedure in which plasmid DNA covalently bound to diazotised paper is used to select complementary mRNA sequences, the cloned sequence was shown to hybridise to an mRNA which directed the synthesis of vitellogenin when translated in a reticulocyte lysate cell-free system.     

DNaseI-hypersensitive sites at promoter-like sequences in the spacer of Xenopus laevis and Xenopus borealis ribosomal DNA.

We have detected a DNAseI hypersensitive site in the ribosomal DNA spacer of Xenopus laevis and Xenopus borealis. The site is present in blood and embryonic nuclei of each species. In interspecies hybrids, however, the site is absent in unexpressed borealis rDNA, but is present normally in expressed laevis rDNA. Hypersensitive sites are located well upstream (over lkb) of the pre-ribosomal RNA promoter. Sequencing of the hypersensitive region in borealis rDNA, however, shows extensive homology with the promoter sequence, and with the hypersensitive region in X. laevis. Of two promoter-like duplications in each spacer, only the most upstream copy is associated with hypersensitivity to DNAaseI. Unlike DNAaseI, Endo R. MspI digests the rDNA of laevis blood nuclei at a domain extending downstream from the hypersensitive site to near the 40S promoter. Since the organisation of conserved sequence elements within this "proximal domain" is similar in three Xenopus species whose spacers have otherwise evolved rapidly, we conclude that this domain plays an important role in rDNA function.     

Mitochondrial DNA in Xenopus laevis oocytes. I. Displacement loop occurrence

Displacement loops are found in mitochondrial DNA isolated from the ovaries of mature female Xenopus laevis. These displacement loops subtend some 7% of the contour length of a mitochondrial circular DNA. When mature oocytes are shed as unfertilized eggs at least 76% of the mitochondrial DNA in these eggs contains displacement loops. The implications of these findings are discussed with respect to displacement loop occurrence in other mitochondrial DNAs and especially with respect to mitochondrial DNA replication.     

Patterns of intra- and interarray sequence variation in alpha satellite from the human X chromosome: evidence for short-range homogenization of tandemly repeated DNA sequences

A number of processes, such as sequence conversion, unequal crossingover, and molecular drive, have been postulated to explain the homogenization of tandemly repeated DNA families. To investigate the nature and extent of such processes in the alpha satellite family of centromeric DNA, we determined the nucleotide sequence of approximately 700 bp from each of 40 representative alpha satellite repeats from six sources of human X chromosomes, obtaining a total of approximately 28 kb of sequence data. Sequence divergence among the repeats examined was low, with an average pairwise difference of approximately 1%. Pairwise comparisons of all repeats indicate that the degree of similarity for those repeats in physical proximity (within approximately 15 kb) of each other is significantly greater than that for randomly located repeats, from either the same or different X chromosomes, suggesting that the mechanisms predicted to homogenize these arrays are effectively short-range in action. Analysis of individual patterns of sequence variation allows the assignment of haplotypes for five high-copy-number diagnostic positions and reveals distinct positions of equilibrium and disequilibrium within the repeat. These analyses address hypotheses about the origin of the observed patterns of variation throughout alpha satellite evolution.     

Tandemly repeated sequences in the mitochondrial DNA control region and phylogeography of the Pike-Perches Stizostedion

DNA sequences from the mitochondrial DNA control region are used to test the phylogeographic relationships among the pike-perches, Stizostedion (Teleostei: Percidae) and to examine patterns of variation. Sequences reveal two types of variability: single nucleotide polymorphisms and 6 to 14 copies of 10- to 11-base-pair tandemly repeated sequences. Numbers of copies of the tandem repeats are found to evolve too rapidly to detect phylogenetic signal at any taxonomic level, even among populations. Sequence similarities of the tandem repeats among Stizostedion and other percids suggest concerted evolutionary processes. Predicted folding of the tandem repeats and their proximity to termination-associated sequences indicate that secondary structure mediates slipped-strand mispairing among the d-loop, heavy, and light strands. Neighbor-joining and maximum parsimony analyses of sequences indicate that the genus is divided into clades on the continents of North America and Eurasia. Calibrating genetic distances with divergence times supports the hypothesis that Stizostedion dispersed from Eurasia to North America across a North Pacific Beringial land bridge approximately 4 million years before present, near the beginning of the Pliocene Epoch. The North American S. vitreum and S. canadense appear separated by about 2.75 million years, and the Eurasian S. lucioperca and S. volgensis are diverged by about 1.8 million years, suggesting that speciation occurred during the late Pliocene Epoch.     

Chromosome localization and orientation of the simple sequence repeat of human satellite I DNA

The predominant chromosomal locations of human satellite I DNA were detected using fluorescent in situ hybridization (FISH). Synthetic deoxyoligonucleotides designed from consensus sequences of the simple sequence repeats of satellite 1 were used as probes. The most abundant satellite I repeat, the-A-B-A-B-A-form, is located at the pericentromeric regions of chromosomes 3, 4, 13, 14, 15, 21, and 22. The less abundant-B-B-B-form was not detected on chromosome 4, but was present at all the other locations. A variation of FISH that allows strand-specific hybridization of single-stranded probes (CO-FISH) determined that the human satellite I sequences are predominantly arranged in head-to-tail fashtion along the DNA strand.