Structural exon database, SEDB, mapping exon boundaries on multiple protein structures

SUMMARY: Comparative analysis of exon/intron organization of genes and their resulting protein structures is important for understanding evolutionary relationships between species, rules of protein organization and protein functionality. We present Structural Exon Database (SEDB), with a Web interface, an application that allows users to retrieve the exon/intron organization of genes and map the location of the exon boundaries and the intron phase onto a multiple structural alignment. SEDB is linked with Friend, an integrated analytical multiple sequence/structure viewer, which allows simultaneous visualization of exon boundaries on structure and sequence alignments. With SEDB researchers can study the correlations of gene structure with the properties of the encoded three-dimensional protein structures across eukaryotic organisms. AVAILABILITY: SEDB is publicly available at http://glinka.bio.neu.edu/SEDB/SEDB.html SUPPLEMENTARY INFORMATION: On the SEDB Web site.     

The Exon/Intron Organization and Chromosomal Mapping of the Mouse ADAMTS-1 Gene Encoding an ADAM Family Protein with TSP Motifs

ADAM is a recently discovered gene family that encodes proteins with a disintegrin and metalloproteinase. ADAMTS-1 is a gene encoding a new member protein of the ADAM family with the thrombospondin (TSP) type I motif, the expression of which is associated with inflammatory processes. In the present study, we have characterized the exon/intron organization of the mouse ADAMTS-1 gene. The ADAMTS-1 gene is composed of nine exons, all of which are present within the 9.2-kb genomic region. Among the nine exons, exons 1, 5, and 6 encode a proprotein domain, a disintegrin-like domain, and a TSP type I motif, respectively, of the ADAMTS-1 protein, suggesting that there is a correlation between exon/intron organization and functional domains. In addition, the exon/ intron organization of the ADAMTS-1 gene is very different from that of the metalloproteinase-like/disintegrin-like/cysteine-rich protein gene (MDC) (ADAM11), suggesting that the genomic structure of ADAM family genes is not necessarily conserved. Furthermore, fluorescencein situhybridization revealed that the ADAMTS-1 gene is located in region C3–C5 of chromosome 16, to which none of the previously identified ADAM genes have been mapped.     

Origin of structural domains of the serum-albumin gene family and a predicted structure of the gene for vitamin D-binding protein

We have recently determined complete DNA sequences for the human albumin and alpha-fetoprotein [AFP] genes and thus have identified their detailed structures. Each is composed of three domains of four exons, three of which are internal and one of which is a domain-linking exon. Equivalent exons in each domain show sufficient sequence and structural similarity to be considered homologous; additional unique exons at each end of the gene show no similarity to the internal triplicated structures. Since earlier, conflicting evolutionary models were based on analysis of single gene structures, we derived from five genes a series of consensus sequences representing the three internal exons as well as the domain-linking exon. The five genes were human and rat albumin and human, mouse, and rat AFP genes. Structurally equivalent exons of the different domains are shown to have arisen from a single exon in a one-domain precursor. Exons that bridge the domains arose from an unequal crossover that fused two exons of the precursor. Our model suggests that part of the coding sequence of the one-domain precursor may have been derived from an intron, by way of loss of a splice site. The consensus sequences were used to propose an intron-exon structure for the related gene encoding the serum vitamin D-binding protein (DBP). DBP is truncated relative to albumin and AFP, and we submit that this results from deletion of two internal exons in the third domain of the gene rather than from premature termination of the coding sequence.     

Two homologous genes, originated by duplication, encode the human hnRNP proteins A2 and A1.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 belongs, with A1, B1 and B2, to the basic protein subset of the hnRNP complex in mammalian cells. All these proteins share a modular structure consisting of two conserved RNA binding domains linked to less conserved Gly-rich domains (2xRBD-Gly). In the framework of our studies on the genetic basis of hnRNP proteins structure and diversity we have isolated and sequenced the A2 gene and compared it to the previously described A1 gene. The A2 gene, which exists in a single copy on Ch. 7 band p15, is split in 12 exons including an alternatively spliced 36 nt mini exon specific for the human hnRNP protein B1. In this work we show that the intron/exon organisation of the A2 gene is identical to that of the A1 gene over the entire length, indicating a common origin by gene duplication. Moreover the comparison of corresponding exons evidences significant conservation also in the apparently divergent Gly-rich domains that could define previously unenvisaged structural and/or functional motifs. The A2 gene promoter is also analysed in comparison to that of the A1 gene.     

Ovoinhibitor introns specify functional domains as in the related and linked ovomucoid gene

We have isolated cDNA clones and determined the gene structure of chicken ovoinhibitor, a seven domain Kazal serine proteinase inhibitor. Using RNA blot hybridization analysis, the gene was identified initially as a region 9-23 kilobases upstream of the gene for the related inhibitor ovomucoid. Ovoinhibitor RNA appears in oviduct and liver. cDNA clones were identified by screening an oviduct cDNA library with a nick-translated DNA restriction fragment which contained an exon of the gene. The mature protein sequence derived from a cDNa clone is in excellent agreement with that which we obtained from direct sequencing of purified ovoinhibitor. The protein-sequencing strategy is reported. The P1 amino acids of the Kazal domains are consistent with the known broad inhibitory specificity of ovoinhibitor. The gene is about 10.3 kilobases in length and consists of 16 exons. Each Kazal domain is encoded by two exons. Like ovomucoid, introns fall between the coding sequences of the ovoinhibitor domains, an arrangement which may have facilitated domain duplication. The intradomain intron occurs in an identical position in all of the ovoinhibitor and ovomucoid Kazal domains, suggesting that this intron was present in the primordial inhibitor gene. We discuss the location of the intradomain intron in relation to the known structure of four Kazal inhibitors and suggest a scheme for the evolution of the ovoinhibitor gene.     

A genetic polymorphism in a functional domain of human pregnancy zone protein: the bait region. Genomic structure of the bait domains of human pregnancy zone protein and alpha 2 macroglobulin.

Genomic clones containing the exons coding for the bait domain of human pregnancy zone protein and alpha 2 macroglobulin were isolated and fragments containing the bait exons were sequenced. It is shown that the bait domains of both alpha 2 macroglobulin and pregnancy zone protein are encoded by two exons, with conserved exon/intron boundaries. A genetic polymorphism showing either a Met or a Val residue as the sixth amino acid of the pregnancy zone protein bait domain was detected with the rare Met allele showing a gene frequency of 0.065.     

Characterization of the exon structure of the Menkes disease gene using vectorette PCR

The gene defective in Menkes disease, an X-linked recessive disturbance of copper metabolism, has been isolated and predicted to encode a copper-binding P-type ATPase. We determined the complete exon—intron structure of the Menkes disease gene, which spans about 150 kb of genomic DNA. The gene contains 23 exons, and the ATG start codon is in the second exon. All of the exon—intron boundaries were sequenced and conformed to the GT/AT rule, except for the 5′ splice site of intron 9. A preliminary comparison demonstrated a striking similarity between the exon structures of the Menkes and Wilson disease genes, giving insight into their evolution.     

Intron-dependent evolution of the nucleotide-binding domains within alcohol dehydrogenase and related enzymes.

It has been suggested that the intron/exon structure of a gene corresponds to its evolutionary history. Accordingly, early in evolution DNA segments encoding short functional polypeptides may have been rearranged (exon-shuffling) to create full-length genes and RNA splicing may have been developed to remove intervening sequences (introns) in order to preserve translational reading frames. A conflicting viewpoint would be that introns were randomly inserted into previously uninterrupted genes after their initial evolutionary development. If so, the sites of introns would be unlikely to consistently reflect the domain structure of the protein. To address this question, the intron/exon structure of the gene encoding human alcohol dehydrogenase (ADH) was determined and compared to the gene structures for other ADHs and related proteins, all of which possess nucleotide-binding domains. Our results indicate that the introns in the nucleotide-binding domains of all the genes examined do indeed fall at positions which separate the short functional polypeptides (i.e. beta strands) which are believed to comprise this domain. We argue that our data is most easily explained by the hypothesis that introns were present in an ancestral nucleotide-binding domain which was later rearranged by exon-shuffling to form the various dehydrogenases and kinases which utilize such a domain.     

Organization of the gene encoding the human macrophage mannose receptor (MRC1).

The gene for the human macrophage mannose receptor (MRC1) has been characterized by isolation of clones covering the entire coding region. Sequence analysis reveals that the gene is divided into 30 exons. The first three exons encode the signal sequence, the NH2-terminal cysteine-rich domain, and the fibronectin type II repeat, while the final exon encodes the transmembrane anchor and the cytoplasmic tail. The intervening 26 exons encode the eight carbohydrate-recognition domains and intervening spacer elements. However, no simple correlation between intron boundaries and functional carbohydrate-recognition domains is apparent. The pattern of intron positions as well as comparison of the sequences of the carbohydrate-recognition domains suggests that the duplication of these domains was an evolutionarily ancient event.     

Two exons encode the calmodulin-binding domain in the mouse phosphorylase kinase catalytic subunit gene

The catalytic subunit, γ, of phosphorylase kinase contains two calmodulin-binding sequences that define a domain in γ that is homologous to the troponin-C-binding domain in troponin I. The homology is based on both sequence and functional similarities. To account for this homology, it has been proposed that the calmodulin-binding sequences in γ and the troponin-C-binding domain in troponin I have evolved from a common ancestor. We investigated this possibility by comparing the exon structure of the γ gene with that of the troponin-I gene over their homologous domains. In the quail troponin-I gene, it is known that the entire troponin-C-binding domain is encoded by a single exon. However, two exons are found to encode the calmodulin-binding domain in the γ gene from mouse. This result indicates that convergent evolution may be responsible for the sequence and functional similarities between the homologous domains in troponin I and γ.     

Isolation of the osteonectin gene: evidence that a variable region of the osteonectin molecule is encoded within one exon

A complementary DNA clone for bovine osteonectin was used to isolate the osteonectin gene from two libraries of bovine genomic DNA fragments. Two overlapping clones were obtained whose relationship was determined by restriction mapping and sequence analysis. The two clones contain the entire osteonectin coding region spanning approximately 11 kilobases of genomic DNA. The coding region of the gene was determined, by electron microscopy and DNA sequencing, to reside in nine exons. In addition, there is at least one 5' exon interrupted by an intron in the 5'-nontranslated sequence of the gene. Excluding this 5' exon and the 3'-terminal exon, the exons are small and approximately uniform in size, averaging 130 +/- 17 base pairs. Three of the exons at the 5' end of the gene were sequenced and appear to encode discrete protein domains. For example, the putative exon 2 contains the coding region for the leader peptide of the molecule. The amino-terminal protein sequence was determined for osteonectin extracted from human, rabbit, and chicken bone and compared with those for bovine, mouse, and pig osteonectin. These data suggest that osteonectin is highly conserved between species, interspecies changes being seen primarily at the amino terminus of the protein and specifically in the region encoded by putative exon 3 in the bovine gene.     

Genomic organization of the human CD5 gene

CD5 is a member of the family of receptors which contain extracellular domains homologous to the type I macrophage scavenger receptor cysteine-rich (SRCR) domain. Here, we compare the exon/intron organization of the human CD5 gene with its mouse homologue, as well as with the human CD6 gene, the closest related member of the SRCR superfamily. The human CD5 gene spans about 24.5 kb and consists of at least 11 exons. These exons are conserved in size, number, and structure in the mouse CD5 homologue. No evidence for the biallelic polymorphism reported in the mouse could be found among a population of 100 individuals of different ethnic origins. The human CD5 gene maps to the Chromosome (Chr) 11q12.2 region, 82 kb downstream from the human CD6 gene, in a head-to-tail orientation, a situation which recalls that reported at mouse Chr 19. The exon/intron organization of the human CD5 and CD6 genes was very similar, differing in the size of intron 1 and the number of exons coding for their cytoplasmic regions. While several isoforms, resulting from alternative splicing of the cytoplasmic exons, have been reported for CD6, we only found evidence of a cytoplasmic tailless CD5 isoform. The conserved structure of the CD5 and CD6 loci, both in mouse and human genomes, supports the notion that the two genes may have evolved from duplication of a primordial gene. The existence of a gene complex for the SRCR superfamily on human Chr 11q (and mouse Chr 19) still remains to be disclosed.     

Proteins, exons and molecular evolution

The discovery of the eukaryotic gene structure has prompted research into the potential relationship between protein structure and function and the corresponding exon/intron patterns. The exon shuffling hypothesis put forward by Gilbert and Blake suggests the encodement of structural and functional protein elements by exons which can recombine to create novel proteins. This provides an explanation for the relatively rapid evolution of proteins from a few primordial molecules. As the number of gene and protein structures increases, evidence of exon shuffling is becoming more apparent and examples are presented both from modern multi-domain proteins and ancient proteins. Recent work into the chemical properties and catalytic functions of RNA have led to hypotheses based upon the early existence of RNA. These theories suggest that the split gene structure originated in the primordial soup as a result of random RNA synthesis. Stable regions of RNA, or exons, were utilised as primitive enzymes. In response to selective pressures for information storage, the activity was directly transferred from the RNA enzymes or ribozymes, to proteins. These short polypeptides fused together to create larger proteins with a wide range of functions. Recent research into RNA processing and exon size, discussed in this review, provides a clearer insight into the evolutionary development of the gene and protein structure.     

Structure of the murine complement factor H gene

Factor H is a regulatory protein of the alternative pathway of complement activation comprised of 20 tandem repeating units of 60 amino acids each. A factor H cDNA clone was used to identify 17 genomic clones from a cosmid library. Four clones were selected for analysis of intron/exon junctions and 5' and 3' regions of the gene and for mapping of the exons. The factor H gene was found to be comprised of 22 exons. Each repeating unit is encoded by one exon, except the second repeat, which is coded by two exons; the leader sequence is encoded by a separate exon. The exons range in size from 77 to 210 base pairs (bp) and average 178 bp. They span a region of approximately 100 kilobases (kb) on chromosome 1. The leader sequence exon is 26 kb upstream of the first repeat exon, representing the largest intron. The other introns range in size from 86 bp to 12.9 kb, and the average intron size is 4.7 kb. Analysis of the genomic organization of the factor H gene has provided insight into the protein structure and will enable the construction of deletion mutants for functional studies.     

Massive expansions of Dscam splicing diversity via staggered homologous recombination during arthropod evolution

The arthropod Down syndrome cell adhesion molecule (Dscam) gene can generate tens of thousands of protein isoforms via combinatorial splicing of numerous alternative exons encoding immunoglobulin variable domains organized into three clusters referred to as the exon 4, 6, and 9 clusters. Dscam protein diversity is important for nervous system development and immune functions. We have performed extensive phylogenetic analyses of Dscam from 20 arthropods (each containing between 46 and 96 alternative exons) to reconstruct the detailed history of exon duplication and loss events that built this remarkable system over 450 million years of evolution. Whereas the structure of the exon 4 cluster is ancient, the exon 6 and 9 clusters have undergone massive, independent expansions in each insect lineage. An analysis of nearly 2000 duplicated exons enabled detailed reconstruction of the timing, location, and boundaries of these duplication events. These data clearly show that new Dscam exons have arisen continuously throughout arthropod evolution and that this process is still occurring in the exon 6 and 9 clusters. Recently duplicated regions display boundaries corresponding to a single exon and the adjacent intron. The boundaries, homology, location, clustering, and relative frequencies of these duplication events strongly suggest that staggered homologous recombination is the major mechanism by which new Dscam exons evolve. These data provide a remarkably detailed picture of how complex gene structure evolves and reveal the molecular mechanism behind this process.