Investigating the mechanism of chromosomal deletion: characterization of 39 deletion breakpoints in introns 47 and 48 of the human dystrophin gene

The region of the dystrophin gene containing introns 45-50 is characterized by a high rate of recombination events that give rise to large deletions causing dystrophinopathy. The nucleotide sequence of this intronic region has recently been released in GenBank. With the aim of further understanding the mechanism favoring the occurrence of these deletions, we have characterized the distribution of introns 47 and 48 deletion endpoints in 39 dystrophinopathy patients. In 14 of these patients we were able to sequence the break junction. On these sequences we were able to identify several intronic motifs that could predispose to DNA double-strand breaks. Our results, combined with other literature data, show that unequal homologous recombination is a very poorly represented event in the dystrophin gene, whereas junction features are suggestive of a model of recombination in which DNA double-strand breaks are incorrectly repaired by a nonhomologous end-joining mechanism. The correlation among recombination rate, deletion frequency, and percentage of repetitive elements is discussed.     

A comparative genomic analysis of the cow,pig, and human CFTR genes identifies potential intronic regulatory elements

The identification of sequences within noncoding regions of genes that are conserved between several species may indicate potential regulatory elements. This is important for genes with complex control mechanisms such as the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR demonstrates similar patterns of temporal and spatial expression in human and sheep, but these differ significantly in mouse cftr. The complete sheep CFTR sequence is unavailable so we annotated BAC clones encompassing the CFTR gene from two other artiodactyl species (cow and pig) for comparative sequence analysis. Regions of introns 2, 3, 10, 17a, 18, and 21 and 3' flanking sequence corresponding to human CFTR DNase I hypersensitive sites (DHS) showed high homology in the cow and pig. Cross-species sequence conservation also enabled finer mapping of other human DHS, including those in introns 1, 16, and 20. Additional potential regulatory elements not associated with human DHS were also identified.     

Numerous group I introns with variable distributions in the ribosomal DNA of a lichen fungus.

The length of the small subunit ribosomal DNA (SSU rDNA) differs significantly among individuals from natural populations of the ascomycetous lichen complex Cladonia chlorophaea. The sequence of the 3' region of the SSU rDNA from two individuals, chosen to represent the shortest and longest sequences, revealed multiple insertions within a region that otherwise aligned with a 520-nucleotide sequence of the SSU rDNA in Saccharomyces cerevisiae. The high degree of variability in SSU rDNA size can be accounted for by different numbers of insertions; one individual had two group I introns and the second had five introns, two of which were clearly related to introns at identical positions in the other individual. Yet, introns in different positions, whether within an individual or between individuals, were not similar in sequence. The distribution of introns at three of the positions is consistent with either intron loss or acquisition, and clearly indicates the dynamic variability in this region of the nuclear genome. All seven insertions, which ranged in size from 210 to 228 nucleotides, had the conserved sequence and secondary structural elements of group I introns. The variation in distribution and sequence of group I introns within a short highly conserved region of rDNA presents a unique opportunity for examining the molecular evolution and mobility of group I introns within a systematics framework.     

Birth of new spliceosomal introns in fungi by multiplication of introner-like elements

Spliceosomal introns are noncoding sequences that separate exons in eukaryotic genes and are removed from pre-messenger RNAs by the splicing machinery. Their origin has remained a mystery in biology since their discovery because intron gains seem to be infrequent in many eukaryotic lineages. Although a few recent intron gains have been reported, none of the proposed gain mechanisms can convincingly explain the high number of introns in present-day eukaryotic genomes. Here we report on particular spliceosomal introns that share high sequence similarity and are reminiscent of introner elements. These elements multiplied in unrelated genes of six fungal genomes and account for the vast majority of intron gains in these fungal species. Such introner-like elements (ILEs) contain all typical characteristics of regular spliceosomal introns (RSIs) but are longer and predicted to harbor more stable secondary structures. However, dating of multiplication events showed that they degenerate in sequence and length within 100,000 years to eventually become indistinguishable from RSIs. We suggest that ILEs not only account for intron gains in six fungi but also in ancestral eukaryotes to give rise to most RSIs by a yet unknown multiplication mechanism.     

Bacteria and Archaea Group II introns: additional mobile genetic elements in the environment

Self-splicing group II introns are present in the organelles of lower eukaryotes, plants and Bacteria and have been found recently in Archaea. It is generally accepted that group II introns originated in bacteria before spreading to mitochondria and chloroplasts. These introns are thought to be related to the progenitors of spliceosomal introns. Group II introns are also mobile genetic elements. In bacteria, they appear to spread using either other mobile genetic elements or low-expression regions as target sites. Bacteria and Archaea genome sequence annotations have revealed the diversity of group II intron classes and that they are involved in vertical and horizontal inheritance.     

Retroviral long terminal repeat is the promoter of the gene encoding the tumor-associated calcium-binding protein oncomodulin in the rat

Oncomodulin is a small calcium-binding protein normally found only in extra-embryonic tissues such as the placenta, but whose presence in a variety of tumors has been documented. We have isolated the oncomodulin gene from a Buffalo rat genomic library. The rat gene is approximately 9000 bases in length and consists of five exons and four introns. The introns interrupt the coding sequence of oncomodulin in positions identical with those previously reported for the parvalbumin gene, indicating that the two genes are derived from a common ancestor. Analysis of the promoter sequence of the oncomodulin gene revealed that the gene is under the control of a solo long terminal repeat element related to intracisternal-A particles, a family of endogenous retroviral elements. This represents a unique example of a mammalian gene transcribed in normal and tumor cells, from a promoter of viral origin.     

Intron/exon structure of the chicken pyruvate kinase gene

The chicken pyruvate kinase gene is interrupted by at least ten introns, including nine introns within the coding region. We compare the structure of this gene with the three-dimensional protein structure of the homologous cat muscle enzyme. The introns are not randomly placed--they divide the coding sequence into fairly uniformly sized pieces encoding discrete elements of secondary structure. The introns tend to fall at interruptions between stretches of alpha-helix or beta-sheet residues, and each of the six exons that contribute to the barrel-shaped central domain include one or two repeats of a simple unit, an alpha-helix plus a beta strand. This structure suggests that introns were not inserted into a previously uninterrupted coding sequence, but instead are products of the evolution of the first pyruvate kinase gene. We have found some sequence homology between a segment of pyruvate kinase and the structurally homologous mononucleotide binding fold of alcohol dehydrogenase. The superposition of these two regions aligns an intron from the maize alcohol dehydrogenase gene four nucleotides from an intron in the chicken pyruvate kinase gene.     

RET rearrangements in radiation-induced papillary thyroid carcinomas: high prevalence of topoisomerase I sites at breakpoints and microhomology-mediated end joining in ELE1 and RET chimeric genes

Children exposed to radioactive iodine after the Chernobyl reactor accident frequently developed papillary thyroid carcinomas (PTC). The predominant molecular lesions in these tumors are rearrangements of the RET receptor tyrosine kinase gene. Various types of RET rearrangements have been described. More than 90% of PTC with RET rearrangement exhibit a PTC1 or PTC3 type of rearrangement with an inversion of the H4 or ELE1 gene, respectively, on chromosome 10. To obtain closer insight into the mechanisms underlying PTC3 inversions, we analyzed the genomic breakpoints of 22 reciprocal and 4 nonreciprocal ELE1 and RET rearrangements in 26 post-Chernobyl tumor samples. In contrast to previous assumptions, an accumulation of breakpoints at the two Alu elements in the ELE1 sequence was not observed. Instead, breakpoints are distributed in the affected introns of both genes without significant clustering. When compared to the corresponding wildtype sequences, the majority of breakpoints (92%) do not contain larger deletions or insertions. Most remarkably, at least one topoisomerase I site was found exactly at or in close vicinity to all breakpoints, indicating a potential role for this enzyme in the formation of DNA strand breaks and/or ELE1 and RET inversions. The presence of short regions of sequence homology (microhomologies) and short direct and inverted repeats at the majority of breakpoints furthermore indicates a nonhomologous DNA end-joining mechanism in the formation of chimeric ELE1/Ret and Ret/ELE1 genes.     

Nucleolar introns from Physarum flavicomum contain insertion elements that may explain how mobile group I introns gained their open reading frames.

Comparison of two group I intron sequences in the nucleolar genome of the myxomycete Physarum flavicomum to their homologs in the closely related Physarum polycephalum revealed insertion-like elements. One of the insertion-like elements consists of two repetitive sequence motifs of 11 and 101 bp in five and three copies, respectively. The smaller motif, which flanks the larger, resembles a target duplication and indicates a relationship to transposons or retroelements. The insertion-like elements are found in the peripheral loops of the RNA structure; the positions occupied by the ORFs of mobile nucleolar group I introns. The P. flavicomum introns are 1184 and 637 bp in size, located in the large subunit ribosomal RNA gene, and can be folded into group I intron structures at the RNA level. However, the intron 2s from both P. flavicomum and P. polycephalum contain an unusual core region that lacks the P8 segment. None of the introns are able to self-splice in vitro. Southern analysis of different isolates indicates that the introns are not optional in myxomycetes.     

Relationship of viroids and certain other plant pathogenic nucleic acids to group I and II introns

Summary The nucleotide sequences of viroids contain features believed to be essential for the splicing of group I introns. Common sequence elements include a 16-nucleotide consensus sequence and three pairs of short sequences arranged in the same sequential order in both types of RNAs. The calculated probability of finding sequences resembling the 16-nucleotide consensus sequence in random nucleotide chains showed that at low fidelity (up to 5 mismatched nucleotides), the number of such sequences in viroids, plant viral satellite RNAs, plant viral RNAs and one plant viral DNA, group I introns and flanking exons does not significantly differ from the number expected at random. As the degree of fidelity is increased, the number in both introns and viroids, but not in exons or the other plant pathogens examined, greatly exceeds that expected in random chains. These findings suggest that viroids may have evolved from group I introns and/or that processing of viroid oligomers to monomers may have structural requirements similar to those of group I introns. The nucleotide sequences of viroids do not show close homology with two conserved regions of group II introns, the 14-base pair consensus region and the 5 terminal segment. However, close homology does exist between the conserved sequence of the 3 terminal segment of group II introns and viroids thus suggesting a possible evolutionary or functional relationship.     

Conserved sequences and structures of group I introns: building an active site for RNA catalysis--a review

Group I introns fold to form an active site to mediate their own RNA splicing. Sequence elements conserved among the available set of 66 group I introns are compiled. Comparative sequence analysis leads to the prediction of some conserved structural features that have not been widely appreciated. The possible significance of conserved nucleotides within base-paired duplexes is discussed; they might be involved in base triplets or alternate pairing interactions.     

Mutation of putative branchpoint consensus sequences in plant introns reduces splicing efficiency

Intron lariat formation between the 5' end of an intron and a branchpoint adenosine is a fundamental aspect of the first step in animal and yeast nuclear pre-mRNA splicing. Despite similarities in intron sequence requirements and the components of splicing, differences exist between the splicing of plant and vertebrate introns. The identification of AU-rich sequences as major functional elements in plant introns and the demonstration that a branchpoint consensus sequence was not required for splicing have led to the suggestion that the transition from AU-rich intron to GC-rich exon is a major potential signal by which plant pre-mRNA splice sites are recognized. The role of putative branchpoint sequences as an internal signal in plant intron recognition/definition has been re-examined. Single nucleotide mutations in putative branchpoint adenosines contained within CUNAN sequences in four different plant introns all significantly reduced splicing efficiency. These results provide the most direct evidence to date for preferred branchpoint sequences being required for the efficient splicing of at least some plant introns in addition to the important role played by AU sequences in dicot intron recognition. The observed patterns of 3' splice site selection in the introns studied are consistent with the scanning model described for animal intron 3' splice site selection. It is suggested that, despite the clear importance of AU sequences for plant intron splicing, the fundamental processes of splice site selection and splicing in plants are similar to those in animals.     

The unusual 23S rRNA gene of Coxiella burnetii: two self-splicing group I introns flank a 34-base-pair exon, and one element lacks the canonical omegaG

We describe the presence and characteristics of two self-splicing group I introns in the sole 23S rRNA gene of Coxiella burnetii. The two group I introns, Cbu.L1917 and Cbu.L1951, are inserted at sites 1917 and 1951 (Escherichia coli numbering), respectively, in the 23S rRNA gene of C. burnetii. Both introns were found to be self-splicing in vivo and in vitro even though the terminal nucleotide of Cbu.L1917 is adenine and not the canonical conserved guanine, termed OmegaG, found in Cbu.L1951 and all other group I introns described to date. Predicted secondary structures for both introns were constructed and revealed that Cbu.L1917 and Cbu.L1951 were group IB2 and group IA3 introns, respectively. We analyzed strains belonging to eight genomic groups of C. burnetii to determine sequence variation and the presence or absence of the elements and found both introns to be highly conserved (>/=99%) among them. Although phylogenetic analysis did not identify the specific identities of donors, it indicates that the introns were likely acquired independently; Cbu.L1917 was acquired from other bacteria like Thermotoga subterranea and Cbu.L1951 from lower eukaryotes like Acanthamoeba castellanii. We also confirmed the fragmented nature of mature 23S rRNA in C. burnetii due to the presence of an intervening sequence. The presence of three selfish elements in C. burnetii's 23S rRNA gene is very unusual for an obligate intracellular bacterium and suggests a recent shift to its current lifestyle from a previous niche with greater opportunities for lateral gene transfer.     

Evidence of selection for protein introns in the recAs of pathogenic mycobacteria.

Protein introns are recently discovered genetic elements whose intervening sequences are removed from a precursor protein by an unusual protein splicing reaction. This involves the excision of a central spacer molecule, the protein intron, and the religation of the amino- and carboxy-terminal fragments of the precursor. The recA gene of Mycobacterium tuberculosis contains one such element and we now show that the other major mycobacterial pathogen, Mycobacterium leprae, also possesses a protein intron in its recA, although other mycobacterial recA genes do not. However, these two protein introns are different in size, sequence and location of insertion of their coding sequences into the recAs of M. tuberculosis and M. leprae, indicating that acquisition of the protein introns has occurred independently in the two species, and thus suggesting that there has been selection for splicing in the maturation of RecA in the pathogenic mycobacteria. The M. leprae protein intron provides an example of conditional protein splicing, splicing occurring in M. leprae itself but not when expressed in Escherichia coli, unlike most previously described protein introns. These observations suggest that protein introns may perform a function for their host, rather than being just selfish elements.     

Palindromic repetitive elements in the mitochondrial genome of Volvox

Group I introns were found in the cob and cox I genes of Volvox carteri. These introns contain tandem arrays of short palindromic sequences that are related to each other. Inspection of other regions in the mtDNA revealed that similar palindromic repetitive sequences are dispersed in the non-protein coding regions of the mitochondrial genome. Analysis of the group I intron in the cob gene of another member of Volvocaceae, Volvox aureus, has shown that its sequence is highly homologous to its counterpart in V. carteri with the exception of a cluster of palindromic sequences not found in V. carteri. This indicates that the palindromic clusters were inserted into the introns after divergence of the two species, presumably due to frequent insertions of the palindromic elements during evolution of the Volvocaceae. Possible involvement of the palindromic repetitive elements in the molecular evolution of functional RNAs is discussed.     

The inconsistent distribution of introns in the T-even phages indicates recent genetic exchanges.

Group I self-splicing introns are present in the td, nrdB and sunY genes of bacteriophage T4. We previously reported that whereas the td intron is present in T2, T4 and T6, the nrdB intron is present in T4 only. These studies, which argue in favor of introns as mobile genetic elements, have been extended by defining the distribution of all three T4 introns in a more comprehensive collection of T2, T4 and T6 isolates. The three major findings are as follows: First, all three introns are inconsistently distributed throughout the T-even phage family. Second, different T2 isolates have different intron complements, with T2H and T2L having no detectable introns. Third, the intron open reading frames are inherited or lost as a unit with their respective flanking intron core elements. Furthermore, exon sequences flanking sites where introns are inserted in the T4 td, sunY and nrdB genes were determined for all the different T-even isolates studied. Six of eighteen residues surrounding the junction sequences are identical. In contrast, a comprehensive comparison of exon sequences in intron plus and intron minus variants of the sunY gene indicate that sequence changes are concentrated around the site of intron occurrence. This apparent paradox may be resolved by hypothesizing that the recombination events responsible for intron acquisition or loss require a consensus sequence, while these same events result in sequence heterogeneity around the site.     

Homing endonuclease structure and function

Homing endonucleases are encoded by open reading frames that are embedded within group I, group II and archael introns, as well as inteins (intervening sequences that are spliced and excised post-translationally). These enzymes initiate transfer of those elements (and themselves) by generating strand breaks in cognate alleles that lack the intervening sequence, as well as in additional ectopic sites that broaden the range of intron and intein mobility. Homing endonucleases can be divided into several unique families that are remarkable in several respects: they display extremely high DNA-binding specificities which arise from long DNA target sites (14-40 bp), they are tolerant of a variety of sequence variations in these sites, and they display disparate DNA cleavage mechanisms. A significant number of homing endonucleases also act as maturases (highly specific cofactors for the RNA splicing reactions of their cognate introns). Of the known homing group I endonuclease families, two (HNH and His-Cys box enzymes) appear to be diverged from a common ancestral nuclease. While crystal structures of several representatives of the LAGLIDADG endonuclease family have been determined, only structures of single members of the HNH (I-HmuI), His-Cys box (I-PpoI) and GIY-YIG (I-TevI) families have been elucidated. These studies provide an important source of information for structure-function relationships in those families, and are the centerpiece of this review. Finally, homing endonucleases are significant targets for redesign and selection experiments, in hopes of generating novel DNA binding and cutting reagents for a variety of genomic applications.     

Two human gamma-crystallin genes are linked and riddled with Alu-repeats

A human genomic cosmid clone, pHcos gamma-1, has been isolated containing two closely linked gamma-crystallin genes, oriented in the same direction. The sequence of these genes and their 5' and 3' flanking regions has been determined. The coding regions of both genes are interrupted by two introns. The first introns (94 and 100 bp, respectively) are located in the 5' region of the genes. The second introns (2.82 and 0.95 kb, respectively) divide the genes into two halves, each encoding a structural domain of the gamma-crystallin protein. The coding regions of the two genes show 80% homology. Due to a mutation in the splice acceptor site of the second intron of the first gene, the coding region of its third exon is 3 bp longer than that of the second gene. In the flanking regions several conserved sequence elements were found, including those elements that are known to be necessary for the correct expression of eukaryotic genes. The flanking and intronic regions of the genes contain 'simple sequence' DNA and Alu repeats. The Alu repeats are usually clustered, contain truncated elements, and are often located near simple sequence DNA.