A transposon-like element in the deletion-prone region of the dystrophin gene.

The central portion of the dystrophin gene locus is a preferential site for deletions causing progressive muscular dystrophy of the Duchenne type (DMD). The nucleotide sequence of a deletion junction fragment from a DMD patient was determined, revealing that the proximal breakpoint of the deletion in intron 43 fell within the sequence of a transposon-like element. This segment, belonging to the THE-1 family of human transposable elements, is normally present in a complete form in intron 43 of the dystrophin gene. The deletion mutation was maternally transmitted and eliminated two-thirds of the THE-1 element. Analysis of DNA from additional DMD patients revealed a second deletion with the proximal breakpoint mapping within the same THE-1 element.     

A dimorphic Alu Sb-like insertion in COL3A1 is ethnic-specific

Alu elements are a class of repetitive DNA sequences found throughout the human genome that are thought to be duplicated via an RNA intermediate in a process termed retroposition. Recently inserted Alu elements are closely related, suggesting that they are derived from a single source gene or closely related source genes. Analysis of the type III collagen gene (COL3A1) revealed a polymorphic Alu insertion in intron 8 of the gene. The Alu insertion in the COL3A1 gene had a high degree of nucleotide identity to the Sb family of Alu elements, a family of older Alu elements. The Alu sequence was less similar to the consensus sequence for the PV or Sb2 subfamilies, subfamilies of recently inserted Alu elements. These data support the observations that at least three source genes are active in the human genome, one of which is distinct from the PV and Sb2 subfamilies and predates either of these two subfamilies. Appearance of the Alu insertion in different ethnic populations suggests that the insertion may have occurred in the last 100,000 years. This Alu insert should be a useful marker for population studies and for marking COL3A1 alleles.     

Essential role of duplications of short motif sequences in the genomic evolution of Bombyx mori

Summary The Bombyx fibroin gene has a discrete mosaic structure of various repetitive sequences, which may have evolved through various repeating arrangements. Detailed sequence analysis of the fibroin gene containing coding and noncoding regions revealed that the whole sequence could be arranged as an array of short repetitive sequences. A portion of the intron of the fibroin gene is one of interspersed repetitive elements. We cloned a 1.5-kb DNA fragment of the Bombyx genome that contains interspersed elements homologous to the intron sequence. Sequence comparison between the intron and the 1.5-kb fragment shows that partial duplication has frequently occurred in evolutionary progress, and the resultant repetitive blocks of short motif sequences are abundant in the genome. These facts suggest that tandem duplication of the short motif sequence is an important rearrangement in genomic evolution of the fibroin gene. Offprint requests to: S. Ichimura     

Chloroplast DNA sequences integrated into an intron of a tomato nuclear gene

Summary DNA sequences capable of hybridizing with chloroplast DNA have previously been reported to exist in the nuclear genome of higher plants. Here we show that the third intron of the cultivated tomato (Lycopersicon esculentum) nuclear gene Cab-7, which resides on chromosome 10 and which we recently cloned and sequenced, contains two DNA fragments derived from the coding region of the chloroplast gene psbG. The first fragment, 133 bp long, is located at a site 63 bp from the 3 end of the 833 bp intron. The exact sequence of the 11 nucleotides at the 3 end of the inserting chloroplast sequence is also found at the 5 border of the insertion. A small (107 bp) chloroplast DNA fragment is inserted near the middle of the intron, again with the 3 end of the inserting element (6 bp) duplicated at the 5 border of the insertion. The second insert is a subfragment of the first insert, and is most likely directly derived from it. The psbG insertion sequence was found to be present in the Cab-7 gene of all tomato species examined but not in species from related genera (e.g. Solanum, Petunia, Nicotiana), suggesting that the original transposition event (chloroplast to nucleus) occurred relatively recently-since the divergence of the genus Lycopersicon from other genera in the family Solanaceae, but before radiation of species in that genus.     

IS<Emphasis Type="Italic">Rm31</Emphasis>, a new insertion sequence of the IS<Emphasis Type="Italic">66</Emphasis> family in<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis>

Sinorhizobium meliloti natural populations show a high level of genetic polymorphism possibly due to the presence of mobile genetic elements such as insertion sequences (IS), transposons, and bacterial mobile introns. The analysis of the DNA sequence polymorphism of the nod region of S. meliloti pSymA megaplasmid in an Italian isolate led to the discovery of a new insertion sequence, ISRm31. ISRm31 is 2,803 bp long and has 22-bp-long terminal inverted repeat sequences, 8-bp direct repeat sequences generated by transposition, and three ORFs (A, B, C) coding for proteins of 124, 115, and 541 amino acids, respectively. ORF A and ORF C are significantly similar to members of the transposase family. Amino acid and nucleotide sequences indicate that ISRm31 is a member of the IS66 family. ISRm31 sequences were found in 30.5% of the Italian strains analyzed, and were also present in several collection strains of the Rhizobiaceae family, including S. meliloti strain 1021. Alignment of targets sites in the genome of strains carrying ISRm31 suggested that ISRm31 inserts randomly into S. meliloti genomes. Moreover, analysis of ISRm31 insertion sites revealed DNA sequences not present in the recently sequenced S. meliloti strain 1021 genome. In fact, ISRm31 was in some cases linked to DNA fragments homologous to sequences found in other rhizobia species.     

Analysis of 22 deletion breakpoints in dystrophin intron 49

Over 60% of Duchenne and Becker muscular dystrophies are caused by deletions spanning tens or hundreds of kilobases in the dystrophin gene. The molecular mechanisms underlying the loss of DNA at this genomic locus are not yet understood. By studying the distribution of deletion breakpoints at the genomic level, we have previously shown that intron 49 exhibits a higher relative density of breakpoints than most dystrophin introns. To determine whether the mechanisms leading to deletions in this intron preferentially involve specific sequence elements, we sublocalized 22 deletion endpoints along its length by a polymerase-chain-reaction-based approach and, in particular, analyzed the nucleotide sequences of five deletion junctions. Deletion breakpoints were homogeneously distributed throughout the intron length, and no extensive homology was observed between the sequences adjacent to each breakpoint. However, a short sequence able to curve the DNA molecule was found at or near three breakpoint junctions.     

Is gene deletion in eukaryotes sequence-dependent? A study of nine deletion junctions and nineteen other deletion breakpoints in intron 7 of the human dystrophin gene

Although large deletions comprise 65% of the mutations that underlie most cases of Duchenne and Becker muscular dystrophies, the DNA sequence characteristics of the deletions and the molecular processes leading to their formation are largely unknown. Intron 7 of the human dystrophin gene is unusually large (110 kb) and a substantial number of deletions have been identified with endpoints within this intron. The distribution of 28 deletion endpoints was mapped to local sequence elements by PCR. The break points were distributed among unique sequence, LINE-1, Alu, MIR, MER and microsatellite sequences with frequencies expected from the frequency of those sequences in the intron. Thus, deletions in this intron are not associated primarily with any one of those sequences in the intron. Nine deletion junctions were amplified and sequenced. Eight were deletions between DNA sequences with minimal homology (0–4 bp) and are therefore unlikely to be products of homologous recombination. In the ninth case, a complex rearrangement was found to be consistent with unequal recombinational exchange between two Alu sequences coupled with a duplication. We have hypothesized that a paucity of matrix attachment regions in this very large intron expanded by the insertion of many mobile elements might provoke a chromatin structure that stimulates deletions (McNaughton et al., 1997, Genomics 40, 294–304). The data presented here are consistent with that idea and demonstrate that the deletion sequences are not usually produced by homologous DNA misalignments.     

Structure and evolution of a family of interspersed repetitive DNA sequences inCaenorhabditis elegans

Summary The structure of three members of a repetitive DNA family from the genome of the nematodeCaenorhabditis elegans has been studied. The three repetitive elements have a similar unitary structure consisting of two 451-bp sequences in inverted orientation separated by 491 bp, 1.5 kb, and 2.5 kb, respectively. The 491-bp sequence separating the inverted 451-bp sequences of the shortest element is found adjacent to one of the repeats in the other two elements as well. The combination of the three sequences we define as the basic repetitive unit. Comparison of the nucleotide sequences of the three elements has allowed the identification of the one most closely resembling the primordial repetitive element. Additionally, a process of co-evolution is evident that results in the introduction of identical sequence changes into both copies of the inverted sequence within a single unit. Possible mechanisms are discussed for the homogenization of these sequences. A direct test of one possible homogenization mechanism, namely homologous recombination between the inverted sequences accompanied by gene conversion, shows that recombination between the inverted repeats does not occur at high frequency.     

Transposition of elements of the 412, copia and 297 dispersed repeated gene families in Drosophila.

The stability of elements of three different dispersed repeated gene families in the genome of Drosophila tissue culture cells has been examined. Different amounts of sequences homologous to elements of 412, copia and 297 dispersed repeated gene families are found in the genomes of D. melanogaster embryonic and tissue culture cells. In general the amount of these sequences is increased in the cell lines. The additional sequences homologous to 412, copia and 297 occur as intact elements and are dispersed to new sites in the cell culture genome. It appears that these elements can insert at many alternative sites. We also describe a DNA sequence arrangement found in the D. melanogaster embryo genome which appears to result from a transposition of an element of the copia dispersed repeated gene family into a new chromosomal site. The mechanism of insertion of this copia element is precise to within 90 bp and may involve a region of weak sequence homology between the site of insertion and the direct terminal repeats of the copia element.     

Nucleotide sequence of a gene from Arabidopsis thaliana encoding a myb homologue

A gene encoding a proto-oncogene, a myb-related gene named Atmyb1, was cloned from Arabidopsis thaliana, and its nucleotide sequence was determined. The Atmyb1 gene contains an intron of 494 bp, and there are no highly homologous sequences present in the A. thaliana genome, but evidence was found that other myb-related genes exist. In the 5 flanking region, we found several typical cis-acting elements found in plant promoters. Sequence comparisons revealed that the ATMYB1 protein has a putative DNA-binding domain with two repeats of tryptophan clusters, which is common in MYB-related proteins in plants, while animal MYB-related proteins contain DNA-binding domains with three repeats of tryptophan clusters. The putative DNA-binding domain of the ATMYB1 protein has higher homology with that of the human c-MYB protein than with those of other plant MYB proteins.     

Comparative Sequence Analysis of the <Emphasis Type="Italic">Phytochrome C</Emphasis> Gene and its Upstream Region in Allohexaploid Wheat Reveals New Data on the Evolution of its Three Constituent Genomes

Bread wheat is an allohexaploid with genome composition AABBDD. Phytochrome C is a gene involved in photomorphogenesis that has been used extensively for phylogenetic analyses. In wheat, the PhyC genes are single copy in each of the three homoeologous genomes and map to orthologous positions on the long arms of the group 5 chromosomes. Comparative sequence analysis of the three homoeologous copies of the wheat PhyC gene and of some 5 kb of upstream region has demonstrated a high level of conservation of PhyC, but frequent interruption of the upstream regions by the insertion of retroelements and other repeats. One of the repeats in the region under investigation appeared to have inserted before the divergence of the diploid wheat genomes, but was degraded to the extent that similarity between the A and D copies could only be observed at the amino acid level. Evidence was found for the differential presence of a foldback element and a miniature inverted-repeat transposable element (MITE) 5′ to PhyC in different wheat cultivars. The latter may represent the first example of an active MITE family in the wheat genome. Several conserved non-coding sequences were also identified that may represent functional regulatory elements. The level of sequence divergence (K_s) between the three wheat PhyC homoeologs suggests that the divergence of the diploid wheat ancestors occurred some 6.9 Mya, which is considerably earlier than the previously estimated 2.5–4.5 Mya. K_a/K_s ratios were <0.15 indicating that all three homoeologs are under purifying selection and presumably represent functional PhyC genes. RT-PCR confirmed expression of the A, B and D copies. The discrepancy in evolutionary age of the wheat genomes estimated using sequences from different parts of the genome may reflect a mosaic origin of some of the Triticeae genomes.     

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.     

Structural heterogeneity in the R173 family of rye-specific repetitive DNA sequences

The rye-specific R173 family of repeated DNA sequences consists of ca. 15 000 individual copies per diploid rye (Secale cereale) genome and is distributed over all 7 rye chromosomes in a dispersed manner. Individual R173 elements vary in size between 3 and 6 kb, are generally not arranged as tandem repeats and are flanked by both multi-copy and single-copy sequences. DNA sequence analysis of three R173 elements (R173-1, R173-2 and R173-3) demonstrated a high degree of homology in conserved domains. The structure of R173-1 was quite different from the other two elements: long direct repeats, which represent a rye-specific repetitive sequence, were found at the ends and a 600 bp long domain was replaced by an unrelated sequence of approximately equal size. R173-2 and R173-3 were extremely similar to each other with the exception of a terminal truncation of R173-2. No open reading frames for proteins >20 kDa were present and a database search failed to detect significant homologies to published protein sequences. Despite the transposon like genomic organisation of the R173 family, individual elements lacked sequence features frequently associated with transposons and retrotransposons. In contrast, two of the regions flanking R173 elements showed strong DNA homologies to a 850 bp long region of a proposed wheat retrotransposon and to a 300 bp long region downstream of the wheatGlu-D1 gene.     

Determination of the exon structure of the distal portion of the dystrophin gene by vectorette PCR

The structure of the 3' one-third of the dystrophin gene has not previously been established. We have used vectorette PCR on a yeast artificial chromosome containing part of the human dystrophin gene to determine that there are 20 exons in this region and to characterize adjacent intron sequences of each one. Combined with previous information on the remainder of the gene, this study shows that the coding sequence is distributed between 79 exons. We have used PCR between exons to measure the distances that separate the more closely clustered exons. Vectorette PCR products were used as probes on Southern blots to assign all the 3' exons to genomic HindIII fragments that are commonly detected in the analysis of dystrophin gene deletions. The results will be useful for determining the effect of genomic deletions on the translational reading frame, for setting up genomic PCR assays to confirm point mutations, for analyzing splice site mutations, and for investigating potential cis-acting elements involved in tissue-specific alternative splicing. Vectorette PCR using primers derived from cDNA sequence represents an efficient and widely applicable method for establishing gene structure and obtaining intron sequence flanking exons, starting from a genomic clone and a cDNA sequence.     

The Structure and Organization of the Human Erythroid Anion Exchanger (AE1) Gene

The AE1 (anion exchanger, band 3) protein is expressed in erythrocytes and in the A-type intercalated cells of the kidney distal collecting tubule. In both cell types it mediates the electroneutral transport of chloride and bicarbonate ions across the lipid bilayer, and, in erythrocytes, it also serves as the critical attachment site of the peripheral membrane skeleton. We have characterized the human AE1 gene using overlapping clones isolated from a phage library of human genomic DNA. The gene spans 20 kb and consists of 20 exons separated by 19 introns. The structure of the human AE1 gene corresponds closely with that of the previously characterized mouse AE1 gene, with a high degree of conservation of exon/intron junctions, as well as exon and intron nucleotide sequences. The putative upstream and internal promoter sequences of the human AE1 gene used in erythroid and kidney cells, respectively, are described. We also report the nucleotide sequence of the entire 3′ noncoding region of exon 20, which was lacking in the published cDNA sequences. In addition, we have characterized 9 Alu repeat elements found within the body of the human AE1 gene that are members of 4 related subfamilies that appear to have entered the genome at different times during primate evolution.     

<Emphasis Type="Italic">hATpin</Emphasis>, a Family of MITE-like <Emphasis Type="Italic">hAT</Emphasis> Mobile Elements Conserved in Diverse Plant Species that Forms Highly Stable Secondary Structures

We identified a 178 bp mobile DNA element in lettuce with characteristic CGAGC/GCTCG repeats in the subterminal regions. This element has terminal inverted repeats and 8-bp target site duplications typical of the hAT superfamily of class II mobile elements, but its small size and potential to form a single-stranded stable hairpin-like secondary structure suggest that it is related to MITE elements. In silico searches for related elements identified 252 plant sequences with 8-bp target site duplications and sequence similarity in their terminal and subterminal regions. Some of these sequences were predicted to encode transposases and may be autonomous elements; these constituted a separate clade within the phylogram of hAT transposases. We demonstrate that the CGAGC/GCTCG pentamer maximizes the hairpin stability compared to any other pentamer with the same C + G content, and the secondary structures of these elements are more stable than for most MITEs. We named these elements collectively as hATpin elements because of the hAT similarity and their hairpin structures. The nearly complete rice genome sequence and the highly advanced genome annotation allowed us to localize most rice elements and to deduce insertion preferences. hATpin elements are distributed on all chromosomes, but with significant bias for chromosomes 1 and 10 and in regions of moderate gene density. This family of class II mobile elements is found primarily in monocot species, but is also present in dicot species. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.