Molecular evolution of serpins: homologous structure of the human alpha 1-antichymotrypsin and alpha 1-antitrypsin genes

alpha 1-Antichymotrypsin belongs to a supergene family that includes alpha 1-antitrypsin, antithrombin III, ovalbumin, and angiotensinogen. The human chromosomal alpha 1-antichymotrypsin gene has been cloned and its molecular structure established. The gene is approximately 12 kb in length and contains five exons and four introns. The locations of the introns within the alpha 1-antichymotrypsin gene are identical with those of the human alpha 1-antitrypsin and angiotensinogen genes. Other members of this supergene family contain introns located at nonhomologous positions of the genes. The homologous organization of the alpha 1-antichymotrypsin and alpha 1-antitrypsin genes corresponds with the high degree of homology between their protein sequences and suggests that these loci arose by recent gene duplication. A model is presented for the evolution of both the genomic structure and the protein sequences of the serine protease inhibitor superfamily.     

Isolation of a cDNA clone for human antithrombin III

Antithrombin III (ATIII) is an important plasma protease inhibitor with a central role in the coagulation system. On the basis of its protein sequence, ATIII is one member of a "super family" of protease inhibitors that includes alpha 1-antitrypsin and chicken ovalbumin. An increased risk of thromboembolism is associated with inherited ATIII deficiency. To study the structure and expression of the human ATIII gene, we have isolated complementary (cDNA) clones for ATIII from human liver mRNA. ATIII cDNA clones were identified by hybridization to a mixture of synthetic oligodeoxynucleotides encoding amino acids 251-256 of the ATIII protein sequence. The largest cDNA clone (1.4 kilobases) included the coding region of ATIII mRNA from codon 10 through a 3'-untranslated region. Comparison of ATIII cDNA clones from two different sources revealed a sequence polymorphism at an internal PstI restriction site. Analysis of both total genomic DNAs and an ATIII gene cloned in a bacteriophage Charon 4A showed that the ATIII gene is present once per haploid genome and is distributed over 10-16 kilobases of DNA. Computer-assisted comparison of the cDNA sequence with those for baboon alpha 1-antitrypsin and chicken ovalbumin revealed homologies consistent with their inclusion in the protease inhibitor superfamily.     

Nucleotide sequence and organization of the human S-protein gene: repeating peptide motifs in the "pexin" family and a model for their evolution

The S-protein/vitronectin gene was isolated from a human genomic DNA library, and its sequence of about 5.3 kilobases including the adjacent 5' and 3' flanking regions was established. Alignment of the genomic DNA nucleotide sequence and the cDNA sequence indicated that the gene consisted of eight exons and seven introns. The intron positions in the S-protein gene and their phase type were compared to those in the hemopexin gene which shares amino acid sequence homologies with transin and the S-protein. Three introns have been found at equivalent positions; two other introns are very close to these positions and are interpreted as cases of intron sliding. Introns 3-7 occur at a conserved glycine residue within repeating peptide segments, whereas introns 1 and 2 are at the boundaries of the Somatomedin B domain of S-protein. The analysis of the exon structure in relation to repeating peptide motifs within the S-protein strongly suggests that it contains only seven repeats, one less than the hemopexin molecule. A very similar repeat pattern like that in hemopexin is shown to be present also in two other related proteins, transin and interstitial collagenase. An evolutionary model for the generation of the repeat pattern in the S-protein and the other members of this novel "pexin" gene family is proposed, and the sequence modifications for some of the repeats during divergent evolution are discussed in relation to known unique functional properties of hemopexin and S-protein.     

Organization of two genes encoding cytotoxic T lymphocyte-specific serine proteases CCPI and CCPII

The genes encoding two recently described cytotoxic T cell proteases, CCPI and CCPII, have been isolated and sequenced. The organizations of the coding and noncoding portions of the two genes are very similar to each other and also to the gene encoding rat mast cell protease type II. Similarly to other serine protease genes, each of the active-site residues is contained on a separate exon; however, two introns were found in particularly interesting positions. One occurs within the postulated activation dipeptide and the other in a position close to the active-site Asp residue. This latter intron interrupts the amino acid sequence in the invariant core region of the protein. We believe that these genes represent a new subfamily of serine protease genes.     

Molecular structure and sequence homology of a gene related to alpha 1-antitrypsin in the human genome

A 7.7-kb EcoRI genomic DNA fragment highly homologous to the human alpha 1-antitrypsin (AAT) gene has been cloned. This antitrypsin-related sequence is physically linked to the authentic AAT gene and both are present in a single cosmid clone. Nucleotide sequencing of the AAT-related genomic fragment demonstrated extensive homology with the authentic AAT gene in the introns as well as in the exons. The conservation of all RNA splice sites and lack of internal termination codons in the exonic regions suggest that it may not be a classical pseudogene. If expressed, it could result in a protein of 420 amino acid residues exhibiting a 70% overall homology with human alpha 1-antitrypsin. The signal peptide sequence is well conserved in the related gene, but the active site for protease inhibition of Met-Ser in alpha 1-antitrypsin has been changed to Trp-Ser. These data suggest that the putative protein encoded by the AAT-related gene is a secretory serine protease inhibitor with an altered substrate specificity. Interestingly, even the intronic regions in the related gene exhibit a 65% overall nucleotide sequence homology with those of the authentic AAT gene. These results suggest that the AAT-related gene is derived from a recent duplication of the authentic AAT gene and represents a new member of the serine protease inhibitor superfamily.     

Exon-intron organization of the human gp130 gene

The exon-intron organization and sequences of the exon-intron boundaries of the human gp130 transmembrane receptor gene have been determined using genomic DNAs as samples. The gp130 gene comprises 17 exons and 16 introns. The positions of the exon-intron boundaries show good correlation to the functional/homology regions of gp130. Exons 3-17 code for the gp130 protein, and each subdomain of the receptor is encoded by a set of exons. The coding potential of exons and the intron phasing of the human gp130 gene conform to the patterns observed previously for other cytokine receptor genes. This supports the notions that the gp130 gene evolved from the same ancestral gene that gave rise to other members of the cytokine receptor family.     

A survey of introns in three genes of rotifers

Here we report on the occurrence and position of introns found in three genes of rotifers. A region of the gene for the TATA-box binding protein was examined in three species of Bdelloidea and one of Monogononta. There are two introns in both copies of this gene present in each of the three bdelloids examined – one at a position where introns occur in other eukaryotes and the other at a novel position; the monogonont has no introns in the region examined. A region of the gene encoding the 82 kD heat shock protein was examined in 10 species, with every rotifer class represented. Introns were found in only two species, both bdelloids: one of the species has an intron in all three copies of the gene; the other has an intron in only one of the three copies. Both introns occur at novel positions. The gene for triosephosphate isomerase was examined in one bdelloid. Both copies of the gene in this species contain introns, all at conserved positions: one copy contains five introns, the other copy three. These observations demonstrate the presence of introns in bdelloid rotifers, some in conserved positions, others apparently newly arisen during bdelloid evolution.     

Structural organization of the 5' region of the thyroglobulin gene. Evidence for intron loss and "exonization" during evolution

More than one third of thyroglobulin (1190 residues out of 2750) is made of one peptide motif repeated ten times in tandem. Segments unrelated to the motif interrupt this structure at various places. The corresponding gene region, which extends over 40 x 10(3) bases, was studied in detail. All exon borders and exon/intron junctions were localized precisely and sequenced, and their positions were correlated with the repetitive organization of the protein. When intron positions were compiled on a consensus sequence of all repeats, three categories of introns were observed. Except between repeats numbers 5 and 6, an intron was invariably found within the Cys codon making the limit of each motif. This category of intron most probably reflects the serial duplication events responsible for the evolution of this region of the gene. All other introns, except no. 2, are found at positions were the repetitive structure is disrupted by "inserted" peptides. We present the hypothesis that this second category of introns was already present in the original unit before the first duplication. Thereafter, they would have experienced either complete loss (some units do not contain any intron) or partial or total exonization, resulting in the slipping of intronic material into coding sequence. Intron no. 2, finally, separates motif no. 1 at a position on the boundary between two segments presenting sequence homology. This last type of intron probably reflects an initial duplication event at the origin of a primordial thyroglobulin gene motif. With all these characteristics, the thyroglobulin gene is presented as a paradigm for the analysis of the fate of introns in gene evolution.     

Genomic organization of the human multidrug resistance (MDR1) gene and origin of P-glycoproteins

The MDR1 gene, responsible for multidrug resistance in human cells, encodes a broad specificity efflux pump (P-glycoprotein). P-glycoprotein consists of two similar halves, each half including a hydrophobic transmembrane region and a nucleotide-binding domain. On the basis of sequence homology between the N-terminal and C-terminal halves of P-glycoprotein, we have previously suggested that this gene arose by duplication of a primordial gene. We have now determined the complete intron/exon structure of the MDR1 gene by direct sequencing of cosmid clones and enzymatic amplification of genomic DNA segments. The MDR1 gene includes 28 introns, 26 of which interrupt the protein-coding sequence. Although both halves of the protein-coding sequence are composed of approximately the same number of exons, only two intron pairs, both within the nucleotide-binding domains, are located at conserved positions in the two halves of the protein. The other introns occur at different locations in the two halves of the protein and in most cases interrupt the coding sequence at different positions relative to the open reading frame. These results suggest that the P-glycoprotein arose by fusion of genes for two related but independently evolved proteins rather than by internal duplication.     

Evolution of the fibronectin gene. Exon structure of cell attachment domain

Genomic DNA coding for human fibronectin was identified from a human genomic library by screening with a cDNA clone that specifies the cell attachment domain in human fibronectin. Two clones which together provided more than 22 kilobase pairs of the fibronectin gene were isolated. The exons in this region correspond to approximately 40% of the coding region in the fibronectin gene. They code for the middle region of the polypeptide which consists of homologous repeating segments of about 90 amino acids called type III homologies. Nucleotide sequence of the portion of the gene corresponding to the cell attachment domain showed that the Arg-Gly-Asp-Ser cell attachment site is encoded within a 165-base pair exon. This exon, together with a 117-base pair exon codes for a homology unit. Analysis of the exon/intron organization in some of the neighboring homology units indicated a similar 2-exon structure. An exception to this pattern is that a single large exon codes for a type III homology unit that, due to alternative mRNA splicing, exists in some but not all fibronectin polypeptides. The introns separating the coding sequences for the type III homology units are located in conserved positions whereas the introns that interrupt the coding sequence within the units are in a variable position generating variations in the size of the homologous exons. This exon/intron organization suggests that the type III homology region of the fibronectin gene has evolved by a series of gene duplications of a primordial gene consisting of two exons. Specification of one of these homology units to the cell attachment domain has occurred within this exon/intron arrangement.     

Evolution and structure of the fibrinogen genes: Random insertion of introns or selective loss?

Chromosomal linkage as well as sequence homologies provide unequivocal evidence that the genes for the alpha, beta and gamma chains of fibrinogen arose by successive duplication of a single ancestral gene. Yet, when the three fibrinogen chains are aligned by amino acid homology, the positions of intervening sequences coincide at only two positions for all three chains. While one additional intron occurs at a homologous site in the beta and gamma chains, none of the positions of the remaining 11 introns in the three genes is shared. This arrangement of introns in the three fibrinogen genes suggests that either introns were selectively lost, implying that there is essential information in the retained introns, or the common introns were present in the ancestral fibrinogen gene and introns have been randomly inserted since the triplication of the original gene. The more likely possibility of selective loss of introns implies that the ancestral gene, as it existed about one billion years ago, must have been composed of numerous small exons.     

Carbonic Anhydrase-Related Protein XI: Structure of the Gene in the Greater False Vampire Bat (Megaderma lyra) Compared with Human and Domestic Pig

Carbonic anhydrase-related protein XI (CA-RP XI) is a member of the α-carbonic anhydrase family (encoded by the gene CA-11), which has lost features of the active site required for enzymatic activity. Using PCR, we amplified CA-11 from genomic DNA of the bat Megaderma lyra. To elucidate the gene structure, we sequenced PCR products and compared their sequences with genomic and mRNA sequences known from human and domestic pig. We identified and sequenced eight introns in the bat CA-11. Five introns (introns 3–7) are located in identical or similar positions in other members of the vertebrate α-carbonic anhydrase gene family. Two 5′ introns and one 3′ intron are located in the regions of little or no sequence similarity with other members of the gene family. The low sequence similarity and additional introns suggest a separate evolutionary origin for the 5′ and 3′ portions of the CA-RP XI gene.     

Genomic organization of the serine protease light chain of mouse complement factor I gene

A portion of the mouse complement factor I (mCFI) gene encoding for the mCFI light chain was cloned from a mouse 129/SVJ1 bacterial artificial chromosome library. It contains five exons and four introns. The intron sizes are remarkably different from the human homolog. Several polymorphisms were found in exon 13. One polymorphism was in the coding region, which causes a threonine in the Balb/c mCFI to be replaced by an isoleucine in the 129/SVJ1 mCFI. The other two polymorphisms are located in the 3' untranslated region. The organization of the serine protease domain in mCFI is similar to that of trypsin but very different from that of the other complement serine proteases.     

Organization and evolution of the rat tyrosine hydroxylase gene

This report describes the organization of the rat tyrosine hydroxylase (TH) gene and compares its structure with the human phenylalanine hydroxylase gene. Both genes are single copy and contain 13 exons separated by 12 introns. Remarkably, the positions of 10 out of 12 intron/exon boundaries are identical for the two genes. These results support the idea that these hydroxylase genes are members of a gene family which has a common evolutionary origin. We predict that this ancestral gene would have encoded exons similar to those of TH prior to evolutionary drift to other members of this gene family.     

The genomic structure of the scallop, Patinopecten yessoensis, troponin C gene: a hypothesis for the evolution of troponin C

Two cDNAs encoding troponin C (TnC) isoforms are isolated from the scallop, Patinopecten yessoensis, striated adductor muscle. The sequential differences between these isoforms, named TnC(long) and TnC(short), are restricted in several residues of the C-terminal region. TnC(long) is commonly expressed in both the striated and the smooth adductor muscle; however, TnC(short) is only in the striated adductor muscle. The TnC gene is a single copy gene in the scallop, thus they are expressed through the alternative splicing from the same gene. The scallop TnC gene is constructed from five exons and four introns, and positions of introns are identical with chordate TnC genes, although the scallop TnC possesses no corresponding intron to the fourth intron of chordates. The loss of this intron is also observed in Drosophila TnC; these may be remnants of their ancestor, namely the early metazoan TnC gene might be a five exons-four introns structure. In addition, the absence of the corresponding intron is also observed among protostomian calmodulins (CaMs), a molecule closely related to TnC. This suggests that the common ancestor gene of the TnC superfamily might also be a five exons-four introns structure. Assuming this to be true, the discordance of the fourth intron positions observed among members of the family is well explained by the evolutionary independent gain of the intron on each member's lineage.