Origin of new genes and source for N-terminal domain of the chimerical gene, jingwei, in Drosophila

This paper deals with a general question posed by the origin of new processed chimerical genes: when a new retrosequence inserts into a new genome position, how does it become activated and acquire novel protein function by recruiting new functional domains and regulatory elements? Jingwei (jgw), a newly evolved functional gene with a chimerical structure in Drosophila, provides an opportunity to examine such questions. The source of its exon encoding C-terminal peptide has been identified as an Adh retrosequence, which extends the concept of exon shuffling from recombination to retroposition as a general molecular mechanism for the origin of a new gene. However, the origin of 5' exons remains unclear. We examined two hypotheses concerning the origin of these non-Adh-derived jgw exons: (i) these exons might originate from a unique genomic sequence that fortuitously evolved a standard intron-exon structure and regulatory sequence for jgw; (ii) these exons might be a duplicate of an unrelated previously existing gene. Genomic Southern analysis, in conjunction with construction and screening of a genomic bookshelf (sub-library), was conducted in a group of Drosophila species. The results demonstrated that there are duplicate genes containing the same structure as the recruited portion of jgw. We name this duplicate gene in Drosophila teissieri and Drosophila yakuba and its orthologous gene in Drosophila melanogaster as yellow-emperor (ymp). Thus, the 5' exons/introns originated from a previously existing gene that provided new modules with specific sub-function to create jgw.     

Origin of new genes: evidence from experimental and computational analyses

Exon shuffling is an essential molecular mechanism for the formation of new genes. Many cases of exon shuffling have been reported in vertebrate genes. These discoveries revealed the importance of exon shuffling in the origin of new genes. However, only a few cases of exon shuffling were reported from plants and invertebrates, which gave rise to the assertion that the intron-mediated recombination mechanism originated very recently. We focused on the origin of new genes by exon shuffling and retroposition. We will first summarize our experimental work, which revealed four new genes in Drosophila, plants, and humans. These genes are 10⁶ to 10⁸ million years old. The recency of these genes allows us to directly examine the origin and evolution of genes in detail. These observations show firstly the importance of exon shuffling and retroposition in the rapid creation of new gene structures. They also show that the resultant chimerical structures appearing as mosaic proteins or as retroposed coding structures with novel regulatory systems, often confer novel functions. Furthermore, these newly created genes appear to have been governed by positive Darwinian selection throughout their history, with rapid changes of amino acid sequence and gene structure in very short periods of evolution. We further analyzed the distribution of intron phases in three non-vertebrate species, Drosophila melanogaster, Caenorhabditis elegans, and Arabidosis thaliana, as inferred from their genome sequences. As in the case of vertebrate genes, we found that intron phases in these species are unevenly distributed with an excess of phase zero introns and a significant excess of symmetric exons. Both findings are consistent with the requirements for the molecular process of exon shuffling. Thus, these non-vertebrate genomes may have also been strongly impacted by exon shuffling in general.     

The structure and function of protein modules.

Analysis of protein sequences shows that many proteins in multicellular organisms have evolved by a process of exon shuffling, deletion and duplication. These exons often correspond to autonomously folding protein modules. Many extracellular enzymes have this modular structure; for example, serine proteases involved in blood-clotting, fibrinolysis and complement. The main role of these modules is to confer specificity by protein-protein interactions. Lack of structural information about such proteins has required a new strategy for studying the structure and function of protein modules. The strategy involves the production of individual modules by protein expression techniques, determination of their structure by high resolution nuclear magnetic resonance and definition of functional patches on the modules by site-directed mutagenesis and biological assays. The structures of the growth factor module, the fibronectin type 1 module and the complement module are briefly described. The possible functional roles of modules in various proteins, including the enzymes factor IX and tissue plasminogen activator, are discussed.     

Structure of the human lck gene: differences in genomic organisation within src-related genes affect only N-terminal exons

Although cDNA sequences coding for several Rous sarcoma virus Src-related protein tyrosine kinases (PTKs) have been reported for several years, knowledge of the structure and organisation of genes of the src family is still limited. In this work, a detailed structure and organisation of the human lck gene is reported. A 17-kb genomic clone encoding human p56 Lck, a lymphocyte-specific PTK of the Src-related subfamily, has been isolated. The human lck gene is organized in 13 exons, one more than in the human cellular (c)-src gene. The twelve coding exons are located in this clone, whereas the putative 5'-noncoding exon is probably located very far upstream from the second exon. Splicing sites for exons 4 to 12, which encode both conserved phospholipase-C-like and catalytic domains of the Src-like PTKs, arise exactly at the same position for the human lck, human c-src and c-fgr genes. The only differences concern the splice sites of exons 1' and 2, which encode the unique N-terminal domain of human Lck. These results give further evidence that the different PTKs of the Src-like family have probably evolved through the mechanism of exon shuffling.     

Protein domains correlate strongly with exons in multiple eukaryotic genomes--evidence of exon shuffling?

We conducted a multi-genome analysis correlating protein domain organization with the exon-intron structure of genes in nine eukaryotic genomes. We observed a significant correlation between the borders of exons and domains on a genomic scale for both invertebrates and vertebrates. In addition, we found that the more complex organisms displayed consistently stronger exon-domain correlation, with substantially more significant correlations detected in vertebrates compared with invertebrates. Our observations concur with the principles of exon shuffling theory, including the prediction of predominantly symmetric phase of introns flanking the borders of correlating exons. These results suggest that extensive exon shuffling events during evolution significantly contributed to the shaping of eukaryotic proteomes.     

Evolution of collagen IV genes from a 54-base pair exon: A role for introns in gene evolution

The exon structure of the collagen IV gene provides a striking example for collagen evolution and the role of introns in gene evolution. Collagen IV, a major component of basement membranes, differs from the fibrillar collagens in that it contains numerous interruptions in the triple helical Gly-X-Y repeat domain. We have characterized all 47 exons in the mouse alpha 2(IV) collagen gene and find two 36-, two 45-, and one 54-bp exons as well as one 99- and three 108-bp exons encoding the Gly-X-Y repeat sequence. All these exons sizes are also found in the fibrillar collagen genes. Strikingly, of the 24 interruption sequences present in the alpha 2-chain of mouse collagen IV, 11 are encoded at the exon/intron borders of the gene, part of one interruption sequence is encoded by an exon of its own, and the remaining interruptions are encoded within the body of exons. In such "fusion exons" the Gly-X-Y encoding domain is also derived from 36-, 45-, or 54-bp sequence elements. These data support the idea that collagen IV genes evolved from a primordial 54-bp coding unit. We furthermore interpret these data to suggest that the interruption sequences in collagen IV may have evolved from introns, presumably by inactivation of splice site signals, following which intronic sequences could have been recruited into exons. We speculated that this mechanism could provide a role for introns in gene evolution in general.     

Expansion of protein domain repeats

Many proteins, especially in eukaryotes, contain tandem repeats of several domains from the same family. These repeats have a variety of binding properties and are involved in protein–protein interactions as well as binding to other ligands such as DNA and RNA. The rapid expansion of protein domain repeats is assumed to have evolved through internal tandem duplications. However, the exact mechanisms behind these tandem duplications are not well-understood. Here, we have studied the evolution, function, protein structure, gene structure, and phylogenetic distribution of domain repeats. For this purpose we have assigned Pfam-A domain families to 24 proteomes with more sensitive domain assignments in the repeat regions. These assignments confirmed previous findings that eukaryotes, and in particular vertebrates, contain a much higher fraction of proteins with repeats compared with prokaryotes. The internal sequence similarity in each protein revealed that the domain repeats are often expanded through duplications of several domains at a time, while the duplication of one domain is less common. Many of the repeats appear to have been duplicated in the middle of the repeat region. This is in strong contrast to the evolution of other proteins that mainly works through additions of single domains at either terminus. Further, we found that some domain families show distinct duplication patterns, e.g., nebulin domains have mainly been expanded with a unit of seven domains at a time, while duplications of other domain families involve varying numbers of domains. Finally, no common mechanism for the expansion of all repeats could be detected. We found that the duplication patterns show no dependence on the size of the domains. Further, repeat expansion in some families can possibly be explained by shuffling of exons. However, exon shuffling could not have created all repeats.     

Exon size distribution and the origin of introns

Since it was first recognised that eukaryotic genes are fragmented into coding segments (exons) separated by non-coding segments (introns), the reason for this phenomenon has been debated. There are two dominant theories: that the piecewise arrangement of genes allows functional protein domains, represented by exons, to recombine by shuffling to form novel proteins with combinations of functions; or that introns represent parasitic DNA that can infest the eukaryotic genome because it does not interfere grossly with the fitness of its host. Differing distributions of exon lengths are predicted by these two theories. In this paper we examine distributions of exon lengths for six different organisms and find that they offer empirical evidence that both theories may in part be correct.     

Structure of the gene for human coagulation factor V.

Activated factor V (Va) serves as an essential protein cofactor for the conversion of prothrombin to thrombin by factor Xa. Analysis of the factor V cDNA indicates that the protein contains several types of internal repeats with the following domain structure: A1-A2-B-A3-C1-C2. In this report we describe the isolation and characterization of genomic DNA coding for human factor V. The factor V gene contains 25 exons which range in size from 72 to 2820 bp. The structure of the gene for factor V is similar to the previously characterized gene for factor VIII. Based on the aligned amino acid sequences of the two proteins, 21 of the 24 intron-exon boundaries in the factor V gene occur at the same location as in the factor VIII gene. In both genes, the junctions of the A1-A2 and A2-A3 domains are each encoded by a single exon. In contrast, the boundaries between domains A3-C1 and C1-C2 occur at intron-exon boundaries, which is consistent with evolution through domain duplication and exon shuffling. The connecting region or B domain of factor V is encoded by a single large exon of 2820 bp. The corresponding exon of the factor VIII gene contains 3106 bp. The 5' and 3' ends of both of these exons encode sequences homologous to the carboxyl-terminal end of domain A2 and the amino-terminal end of domain A3 in ceruloplasmin. There is otherwise no homology between the B domain exons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Directed evolution of proteins by exon shuffling

Evolution of eukaryotes is mediated by sexual recombination of parental genomes. Crossovers occur in random, but homologous, positions at a frequency that depends on DNA length. As exons occupy only 1% of the human genome and introns about 24%, by far most of the crossovers occur between exons, rather than inside. The natural process of creating new combinations of exons by intronic recombination is called exon shuffling. Our group is developing in vitro formats for exon shuffling and applying these to the directed evolution of proteins. Based on the splice frame junctions, nine classes of exons and three classes of introns can be distinguished. Splice frame diagrams of natural genes show how the splice frame rules govern exon shuffling. Here, we review various approaches to constructing libraries of exon-shuffled genes. For example, exon shuffling of human pharmaceutical proteins can generate libraries in which all of the sequences are fully human, without the point mutations that raise concerns about immunogenicity.     

Complete structure of the chicken alpha 2(VI) collagen gene

Type VI collagen is a hybrid molecule consisting of a short triple helix flanked by two large globular domains. These globular domains are composed of several homologous repeats which show a striking similarity to the collagen-binding motifs found in von Willebrand factor. The alpha 2(VI) subunit contains three of these homologous repeats termed D1, D2 and D3. We have isolated and characterized the entire gene for chicken alpha 2(VI) collagen. This gene, which is present as a single copy in the chicken genome, is 26 kbp long and comprises 28 exons. All exons can be classified in three groups. (a) The triple-helical domain is encoded by 19 short exons (27-90 bp) separated by introns of phase class 0. These exons are multiples of 9 bp and encode an integral number of collagenous Gly-Xaa-Yaa triplets. (b) The homologous repeats D1-D3 are encoded by one or two very long exons each (153-1578 bp). These exons are separated by introns of phase class 1. (c) The homologous repeats and the collagen sequence are linked to each other by three short adapter segments which are each encoded by a single exon (21-46 bp). The modular nature of the polypeptide is thus clearly reflected by the mosaic structure of its gene. The size of the exons and the phase class of the introns suggest that the alpha 2(VI) gene evolved by duplication and shuffling of two different primordial exons, one of 9 bp encoding a collagen Gly-Xaa-Yaa triplet and one of 600 bp encoding the precursor of the homologous repeats.     

Characterization of the gene for human plasminogen, a key proenzyme in the fibrinolytic system

The organization and structure of the gene coding for plasminogen has been determined by a combination of in vitro amplification of leukocyte DNA from normal individuals and isolation of unique clones from three different human genomic libraries. These clones were characterized by restriction mapping, Southern blotting, and DNA sequencing. The gene for human plasminogen spanned about 52.5 kilobases of DNA and consisted of 19 exons separated by 18 introns. DNA sequence analysis revealed that the five kringle structures in plasminogen were coded by two exons. The nucleotides in the introns at the intron-exon boundaries were GT-AG analogous to those found in other eukaryotic genes. Three polyadenylation sites for plasminogen mRNA were also identified. When the amino acid sequences deduced from the genomic DNA and cDNAs of plasminogen were compared with that of the plasma protein determined by amino acid sequence analysis, an apparent amino acid polymorphism was observed in several positions of the polypeptide chain. Nucleotide sequence analysis of the amplified genomic DNAs and genomic clones also revealed that the plasminogen gene was very closely related to several other proteins, including apolipoprotein(a). This protein may have evolved via duplication and exon shuffling of the plasminogen gene. The presence of another plasminogen-related gene(s) in the human genomic library was also observed.     

Evolutionary fate and expression patterns of chimeric new genes in Drosophila melanogaster

Origin and evolution of new genes contribute a lot to genome diversity. New genes usually form chimeric gene structures through DNA-based exon shuffling and generate proteins with novel functions. We investigated polymorphism of 14 chimeric new genes in Drosophila melanogaster populations and found that eight have premature stop codons in some individuals while six are intact in the population, four of which are under negative selection, suggesting the two evolutionary fates of new chimeric genes after origination: accumulate premature stop codons and pseudolize, or acquire functions and get fixed by natural selection. Different from new genes originated through RNA-based duplication (retroposition) which are usually testis-specific or male-specific expressed, the expression patterns of these new genes through DNA-based exon shuffling are temporally and spatially diverse, implying that they may have the potential to evolve various biological functions despite that they may become pseudogenes or non-protein-coding RNA genes.     

In vitro rapid evolution of fungal immunomodulatory proteins by DNA family shuffling

Fungal immunomodulatory proteins (FIPs) found in a wide variety of mushrooms hold significant therapeutic potential. Despite much research, the structural determinants for their immunomodulatory functions remain unknown. In this study, a DNA shuffling technique was used to create two shuffled FIP protein libraries: an intrageneric group containing products of shuffling between FIP-glu (FIP gene isolated from Ganoderma lucidum) and FIP-gsi (FIP gene isolated from Ganoderma sinense) genes and an intergeneric group containing the products of shuffling between FIP-glu, FIP-fve (FIP gene isolated from Flammulina velutipes), and FIP-vvo (FIP gene isolated from Volvariella volvacea) genes. The gene shuffling generated 426 and 412 recombinant clones, respectively. Using colony blot analysis, we selected clones that expressed relatively high levels of shuffled gene products recognized by specific polyclonal antibodies. We analyzed the DNA sequences of the selected shuffled genes, and testing of their protein products revealed that they maintained functional abilities to agglutinate blood cells and induce cytokine production by splenocytes from Kunming mice in vitro. Meanwhile, the relationships between protein structure and the hemagglutination activity and between the changed nucleotide sites and expression levels were explored by bioinformatic analysis. These combined analyses identified the nucleotide changes involved in regulating the expression levels and hemagglutination activities of the FIPs. Therefore, we were able to generate recombinant FIPs with improved biological activities and expression levels by using DNA shuffling, a powerful tool for the generation of novel therapeutic proteins and for their structural and functional studies.     

Structure and evolution of the bovine prothrombin gene

The cloned bovine prothrombin gene has been characterized by partial DNA sequence analysis, including the 5' and 3' flanking sequences and all the intron-exon junctions. The gene is approximately 15.4 x 10(3) base-pairs in length and comprises 14 exons interrupted by 13 introns. The exons coding for the prepro-leader peptide and the gamma-carboxyglutamic acid-containing region are similar in organization to the corresponding exons in the factor IX and protein C genes. This region has probably evolved as a result of recent gene duplication and exon shuffling events. The exons coding for the kringles and the serine protease region of the prothrombin gene are different in organization from the homologous regions in other genes, suggesting that introns have been inserted into these regions after the initial gene duplication events.