Replicon fusions promoted by the inverted repeats of Tn5. The right repeat is an insertion sequence

Members of three repetitive sequence families were isolated from recombinant λ-genome libraries, and were used to investigate sequence relationships within these families. Studies presented elsewhere show that members of all three repeat sequence families are transcribed tissue-specifically. The thermal stability of intrafamilial heteroduplexes was measured, and the extent of colinearity between related sequences was determined by restriction mapping, heteroduplex visualization, gel blot hybridization, and direct sequencing. One large and very divergent family, named 2108, was shown to consist of an assemblage of many small repeat sequence subfamilies. Each subfamily includes <40 members which are not contiguous in the genome but are very closely related colinear sequence elements several thousand nucleotides in length. The different 2108 subfamilies share only small sequence subelements, which in each subfamily occur in a different linear order and are surrounded by different sequences. A second divergent family consisting of short repetitive sequences, the 2109 family, includes many small internally homologous subfamilies as well. A third family, 2034, displays little internal sequence divergence and no apparent subfamily structure. The repeat sequence subfamilies may be biologically significant units of repetition. Thus specific 2108 subfamilies were shown to be evolutionary conserved to a remarkable degree. Highly homologous 2108 sequences were found shared among sea urchin species which diverged almost 200 million years ago, although only about 10% of the single copy DNA sequences of these species are now homologous enough to crossreact.     

Occurrence of reiterated sequences in an untranslated region of Simian virus 40 DNA determined by nucleotide sequence analysis.

An earlier report (Subramanian, Dhar, and Weissman, 1977c) presented the nucleotide sequence of Eco RII-G fragment of SV40 DNA, which contains the origin of DNA replication. The nucleotide sequence of Eco RII-N fragment located next to Eco RII-G on the physical map of SV40 DNA is presented in this report. Eco RII-N is found to be a tandem duplication of the last 55 nucleotides of Eco RII-G. This tandem repeat is immediately preceded by two other reiterated sequences occurring within Eco RII-G, one of them being a tandem repeat of 21 nucleotides and the other a nontandem repeat of 10 nucleotides. These repetitive sequences occur in close proximity to the origin of DNA replication which is known to contain other specialized sequences such as a few palindromes (one of which is 27 long and possesses a perfect 2-fold axis of symmetry), one "true" palindrome, and a long A/T-rich cluster. The repeats (and the replication origin) occur within an untranslated region of SV40 DNA flanked by (the few) structural genes coding for the "late" proteins on the one side and that (those) coding for the "early" protein(s) on the other side. The reiterated sequences are comparable in some respects to repetitive sequences occurring in eucaryotic DNAs. Possible biological functions of the repeats are discussed.     

TU elements: a heterogeneous family of modularly structured eucaryotic transposons.

We describe here a family of foldback transposons found in the genome of the higher eucaryote, the sea urchin Strongylocentrotus purpuratus. Two major classes of TU elements have been identified by analysis of genomic DNA and TU element clones. One class consists of largely similar elements with long terminal inverted repeats (IVRs) containing outer and inner domains and sharing a common middle segment that can undergo deletions. Some of these elements contain insertions. The second class is highly heterogeneous, with many different middle segments nonhomologous to those of the first-class and variable-sized inverted repeats that contain only an outer domain. The middle and insertion segments of both classes carry sequences that also are found unassociated from the inverted repeats at many other genomic locations. We conclude that the TU elements are modular structures composed of inverted repeats plus other sequence domains that are themselves members of different families of dispersed repetitive sequences. Such modular elements may have a role in the dispersion and rearrangement of genomic DNA segments.     

Characteristics of individual repetitive sequence families in the sea urchin genome studied with cloned repeats

Cloned repetitive sequences from the S. purpuratus genome a few hundred to approximately 1000 nucleotides long were used to investigate the characteristics of individual repetitive sequence families. They were terminally labeled by the kinase procedure and reacted with sheared S. purpuratus DNA. Repetition frequencies were measured for 26 individual families and were found to vary from a few to several thousand copies per genome. Estimates of sequence divergence were made for 18 cloned repeat families by measuring thermal stability of the heteroduplexes formed between the genomic DNA and the cloned fragments, compared with that of the renatured cloned fragments. The difference was <4°C for three of the 18 families, and <10°C for 13 of the 18 families. These 13 repetitive sequence families lack any detectable highly divergent sequence relatives, and the results reported are shown not to change when the renaturation criterion is lowered below 55°C in 0.18 M Na⁺. Five of the 18 cloned families displayed greater sequence divergence. The average sequence divergence of the total short repetitive sequence fraction of S. purpuratus DNA was found to match closely the average of the divergences of the cloned repeat sequences.     

Characterization of long and short repetitive sequences in the sea urchin genome

Long and short repetitive sequences were purified from the DNA of Paracentrotus lividus under conditions designed to optimize the yield of complete, end to end sequences. Double-stranded long repeat DNA prepared in this manner ranged in length from approximately 3000 to 15 000 nucleotide pairs with average sizes of approximately 6000 base pairs. In the electron microscope, long repeat DNA was observed to possess continuous sequences that often appeared to be terminated by one or more loops and/or fold backs. Long repeat DNA sequences, resheared to 300 base pairs, were found to have an average melting point identical to that for sheared native DNA. Thus, the reassociated duplexes of long repetitive DNA seem to possess very few mismatched base pairs. Reassociation kinetic analyses indicate that the majority of the long repeat sequences are reiterated only 4--7 times per haploid amount of DNA. Melt-reassociation analyses of short repetitive DNA, at several criteria, support the previously held concept that these sequences belong the sets or families of sequences which are inexact copies of one another. Our studies also support hypotheses suggesting that short repetitive sequences belong to families which may have arisen via distinct salttatory events. The relationships between long and short repetitive DNA sequences are considered with respect to widely held concepts of their sequence organization, evolution, and possible functions within eucaryotic genomes. A model for the possible organization of short repeats within long repetitive DNA sequences is also presented.     

Structure Analysis of Two <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> and <Emphasis Type="BoldItalic">Neospora caninum</Emphasis> Satellite DNA Families and Evolution of Their Common Monomeric Sequence

A family of repetitive DNA elements of approximately 350 bp—Sat350—that are members of Toxoplasma gondii satellite DNA was further analyzed. Sequence analysis identified at least three distinct repeat types within this family, called types A, B, and C. B repeats were divided into the subtypes B1 and B2. A search for internal repetitions within this family permitted the identification of conserved regions and the design of PCR primers that amplify almost all these repetitive elements. These primers amplified the expected 350-bp repeats and a novel 680-bp repetitive element (Sat680) related to this family. Two additional tandemly repeated high-order structures corresponding to this satellite DNA family were found by searching the Toxoplasma genome database with these sequences. These studies were confirmed by sequence analysis and identified: (1) an arrangement of AB1CB2 350-bp repeats and (2) an arrangement of two 350-bp-like repeats, resulting in a 680-bp monomer. Sequence comparison and phylogenetic analysis indicated that both high-order structures may have originated from the same ancestral 350-bp repeat. PCR amplification, sequence analysis and Southern blot showed that similar high-order structures were also found in the Toxoplasma-sister taxon Neospora caninum. The Toxoplasma genome database ( ) permitted the assembly of a contig harboring Sat350 elements at one end and a long nonrepetitive DNA sequence flanking this satellite DNA. The region bordering the Sat350 repeats contained two differentially expressed sequence-related regions and interstitial telomeric sequences.     

Evolutionary change in the repetition frequency of sea urchin DNA sequences

The frequency of occurrence of particular repetitive sequence families has been estimated in the DNA of the three sea urchin species Strongylocentrotus purpuratus, Strongylocentrotus franciscanus and Lytechinus pictus using individual cloned S. purpuratus repetitive sequence elements. Cloned repetitive sequence elements as described by Scheller et al. (1977a) were prepared by reassociation of S. purpuratus DNA fragments to repetitive Cot, digestion with single-strand-specific nuclease S1 and ligation of synthetic restriction sites to their ends. The sequences were cloned by insertion at the Eco RI site of plasmid RSF2124, labeled, strand-separated and reassociated with 800–900 nucleotide long unlabeled DNA. Both kinetic (genomic DNA excess) and saturation (cloned DNA excess) estimates of frequencies were made. For nine cloned fragments, the ratio of the repetition frequency in S. purpuratus DNA to that in S. franciscanus DNA ranges from about 20 to about 1. In the four cases examined, only a few copies were detected in the DNA of L. pictus. Estimates have also been made of frequency changes in many repetitive families by measuring the reassociation of labeled repetitive DNA fractions of each species with total DNA from other species. In each reciprocal comparison, the labeled repetitive sequences reassociate more slowly with DNA of other species than with DNA of the species from which they were prepared. Thus it appears that the dominant repetitive sequence families in the DNA of each species are present at lower frequencies in the DNA of closely related species. Measurements of thermal stability have been made of S. purpuratus cloned repetitive sequences reassociated with S. franciscanus DNA or S. purpuratus DNA. Most families have changed both in frequency and sequence, although some have changed little in sequence but show great changes in frequency.     

Distinct characteristics of loop sequences of two Drosophila foldback transposable elements.

A few foldback (FB) transposable elements have, between their long terminal inverted repeats, central loop sequences which have been shown to be different from FB inverted repeat sequences. We have investigated loop sequences from two such FB elements by analyzing their genomic distribution and sequence conservation and, in particular, by determining if they are normally associated with FB elements. One of these FB loop sequences seems to be present in a few conserved copies found adjacent to FB inverted repeat sequences, suggesting that it represents an integral component of some FB elements. The other loop sequence is less well-conserved and not usually associated with FB inverted repeats. This sequence is a member of another family of transposable elements, the HB family, and was found inserted in an FB element only by chance. We compare the complete DNA sequences of two HB elements and examine the ends of four HB elements.     

Mobile dispersed genetic element MDG1 of Drosophila melanogaster: nucleotide sequence of long terminal repeats.

Long terminal repeats (LTRs) of two members of mdg1 family were sequenced. In the both cases, they are represented by perfect direct repeats 442 and 444 bp in length. Sixteen nucleotides in the LTRs of two different mdg1 elements are different. Each LTR contains slightly mismatched 16-nucleotide inverted repeats located at the ends of the LTR. Six base pairs closest to the termini of LTR form perfect inverted repeats. On the gene-distal sides of LTRs, short 4-nucleotide direct repeats are located, probably representing the duplication of a target DNA sequence arising from insertion of mdg. They are different in the two cases analyzed. Just as the other analyzed eukaryotic transposable elements, mdg1 starts with TGT and ends with ACA. Within the both strands of LTR, the sequences similar to Hogness box (a putative signal for RNA initiation, or a selector) and AATAAA blocks (putative polyadenylation signals) are present. The LTR of mdg1 contains many short direct and inverted repetitive sequences. These include a 10-nucleotide sequence forming a perfect direct repeat with the first ten nucleotides of the LTR. A region of LTR about 70 bp long is represented by simple repetitive sequences (TAT).     

Tsp transposons: a heterogeneous family of mobile sequences in the genome of the sea urchin Strongylocentrotus purpuratus.

In the preceding paper (J.B. Cohen, B. Hoffman-Liebermann, and L. Kedes, Mol. Cell. Biol., 5:2804-2813, 1985), we described the nucleotide sequence of ISTU4, which is a member of a new family of repetitive sequences, the Tsp family, present in a higher eucaryote, the sea urchin Strongylocentrotus purpuratus. We provided evidence that individual members of this family can act as transposable elements. Here we describe our structural analysis of the Tsp element family, which numbers about 1,000 members per haploid genome. Hybridization and nucleotide sequence analysis of several genomic Tsp clones demonstrate that structurally most Tsp elements resemble ISTU4. Tsp elements range in size up to about 1.3 kilobase pairs, have terminal domains that are conserved between the various examples studied, and contain a central portion of varying size, which may be extensively diverged. Structurally, however, the central portions are very similar and consist of several approximately 150-base-pairs-long, tandemly arranged, imperfect repeats, which are followed by a truncated repeat. The structural analysis is consistent with the possibility that the individual Tsp elements differ by multiples of these 150-base-pair repeats. One variant genomic clone has a solitary repeat and lacks the truncated repeat. The nucleotide sequences of different repeats of a single Tsp element can diverge extensively. The truncated repeat is divergent from most of the repeats, but in one case it is almost identical to a repeat of the same element. Comparison of the sequences from different elements enabled us to determine the boundaries of each structural domain and allows us to propose that each of these domains may be independent units of genetic information. Analysis of the population of Tsp-related sequences in the S. purpuratus genome by genomic blot hybridization suggests that most Tsp family members share the same overall structure. In addition, there is a structural element, about 70 base pairs long, that appears to interrupt the tandem arrangement of the 150-base-pair repeats at regular intervals.     

Long and short repeats of sea urchin DNA and their evolution

Repeated sequences cloned from the DNA of the sea urchin S. purpuratus were used as probes to measure the lengths of individual families of repeats. Some probes reassociated much more rapidly with preparations of long repeats than with short repeats while others reassociated more rapidly with short repeats than with long repeats. In this way two of five cloned repeats were shown to represent families with a great majority of sequences in the long class. One represented a family with similar numbers of long and short class members. Two were members of predominantly short class families. — The cloned repeats representing long class families, formed more precise duplexes than those representing short class families. Thermal stability measurements using S. purpuratus or S. franciscanus driver DNA showed that precise repetitive sequences have as great an interspecies sequence difference as the less precise repeats. Thus the precision of many families may result from recent multiplication rather than from selective pressure on the DNA sequences. Measurements of evolutionary frequency change show a clear correlation between the frequency change and the size of families of repeats in S. purpuratus. Comparison with S. franciscanus indicates that many of the large size families in S. purpuratus are those that have grown in size since these two species diverged.     

A "CAT" family of repetitive DNA sequences in Saccharomyces cerevisiae

An oligonucleotide probe was used to isolate yeast genomic clones containing DNA sequences with repetitive elements consisting primarily of a tandemly arranged trinucleotide, CAT. Hybridization analyses estimate that the yeast genome contains 40-50 CAT clusters, representing the first repetitive DNA sequence family found in yeast. Sequence analyses show short spacers between the CAT repeats consisting of closely related trinucleotides, primarily CGT. Some of the CAT clusters are located in longer repeating elements with lengths of 7 nucleotides or more. In one case a three-times-repeated 27-nucleotide sequence bears striking homology to the 21-base pair repeat region of the mammalian simian virus 40 promoter element. Hybridization studies further suggest that the "CAT" sequences may be widely dispersed in many diverse organisms including Escherichia coli, Drosophila, and man.     

A high frequency of length polymorphisms in repeated sequences adjacent to Alu sequences

We describe a new class of DNA length polymorphism that is due to a variation in the number of tandem repeats associated with Alu sequences (Alu sequence-related polymorphisms). The polymerase chain reaction was used to selectively amplify a (TTA)n repeat identified in the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase gene from genomic DNA of 41 human subjects, and the size of the amplified products was determined by gel electrophoresis. Seven alleles were found that differed in size by integrals of three nucleotides. The allele frequencies ranged from 1.5% to 52%, and the overall heterozygosity index was 62%. The polymorphic TTA repeat was located adjacent to a repetitive sequence of the Alu family. A homology search of human genomic DNA sequences for the trinucleotide TTA (at least five members in length) revealed tandem repeats in six other genes. Three of the six (TTA)n repeats were located adjacent to Alu sequences, and two of the three (in the genes for beta-tubulin and interleukin-1 alpha) were found to be polymorphic in length. Tandemly repetitive sequences found in association with Alu sequences may be frequent sites of length polymorphism that can be used as genetic markers for gene mapping or linkage analysis.     

Structure and unusual characteristics of a new family of transposable elements in the sea urchin Strongylocentrotus purpuratus.

The transposable element family TU of the sea urchin Strongylocentrotus purpuratus, a higher eucaryote, has recently been described (D. Liebermann, B. Hoffman-Liebermann, J. Weinthal, G. Childs, R. Maxson, A. Mauron, S.N. Cohen, and L. Kedes, Nature [London] 306:342-347, 1983). A member of this family, TU4, has an insertion, called ISTU4, of non-TU DNA. ISTU4 is a member of a family of repetitive sequences, which are present in some 1,000 copies per haploid S. purpuratus genome (B. Hoffman-Liebermann, D. Liebermann, L.H. Kedes, and S.N. Cohen, Mol. Cell. Biol. 5:991-1001, 1985). We analyzed this insertion to determine whether it is itself a transposable element. The nucleotide sequence of ISTU4 was determined and showed an unusual structure. There are four, approximately 150 nucleotides long, imperfect direct repeats followed by a single truncated version of these repeats. This region is bounded at either side by approximately 100-nucleotide-long sequences that are not related to each other or to the repeats. Nucleotide sequences at the boundaries of ISTU4-homologous and flanking regions in five genomic clones show that ISTU4 represents a family of sequences with discrete ends, which we call Tsp elements. We showed that the genomic locus that carries a Tsp element in one individual was empty in other individuals and conclude that Tsp elements are a new and different type of transposable element. Tsp elements lack two features common to most other transposable elements: Tsp integration does not result in the duplication of host DNA, and there are no inverted repeats at their termini, although short inverted repeats are present at a distance from the termini.     

The human THE-LTR(O) and MstII interspersed repeats are subfamilies of a single widely distributed highly variable repeat family.

Fifteen examples of the transposon-like human element (THE) LTR and thirteen examples of the MstII interspersed repeat are aligned to generate new consensus sequences for these human repetitive elements. The consensus sequences of these elements are very similar, indicating that they compose subfamilies of a single human interspersed repetitive sequence family. Members of this highly polymorphic repeat family have been mapped to at least 11 chromosomes. Seven examples of the THE internal sequence are also aligned to generate a new consensus sequence for this element. Estimates of the abundance of this repetitive sequence family, derived from both hybridization analysis and frequency of occurrence in GenBank, indicate that THE-LTR/MstII sequences are present every 100-3000 kb in human DNA. The widespread occurrence of members of this family makes them useful landmarks, like Alu, L1, and (GT)n repeats, for physical and genetic mapping of human DNA.     

KpnI families of long, interspersed repetitive DNAs associated with the human β-globin gene cluster

KpnI families of long, interspersed repetitive DNAs are ubiquitous repetitive elements that occur in tens of thousands of copies in primate genomes. KpnI 1.2, 1.5 and two different KpnI 1.8-kb families were found within and flanking a 6.4-kb repeat beginning at 3 kb, 3' from the human β-globin gene. Thus, six different types of KpnI families have now been identified, and four of these are found next to each other in a specific 6.4-kb repeat. Clones of the distinct KpnI families were hybridized to clones of the 6.4-kb repeat and adjacent sequences encompassed within some 17.6 kb of DNA lying 3' to the β-globin gene cluster. The four KpnI families appear to make up the entire length of the 6.4-kb repeat. The linear order of the various cloned KpnI sequences in the repeat is 5'-pBK(1.8)26-pBK.(1.5)54-pBK(1.2)11-pBK(1.8)11-3'. KpnI 1.2-kb sequences were also detected downstream from the 6.4-kb repeat. As in the case of the KpnI 1.2 and 1.5-kb families, the two KpnI 1.8-kb sequence families described here each hybridized with about 15% of all plaques in two independently generated human genome libraries.