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.     

The apolipoprotein(a) gene resides on human chromosome 6q26–27, in close proximity to the homologous gene for plasminogen

Summary Apolipoprotein(a) [apo(a)], the glycoprotein associated with the lipoprotein(a) [Lp(a)] subfraction of plasma lipoproteins, has been shown to exhibit heritable molecular weight isoforms ranging from 400–700 kDa. Increased serum concentrations of Lp(a) correlate positively with the risk of atherosclerosis. Variations in Lp(a) plasma levels among individuals are inherited as a codominant quantitative trait. As part of an effect to define the basis of these variations and further clarify the expression of the protein, we have determined the chromosomal location of the human apo(a) gene. Blot hybridization analysis of DNA from a panel of mouse-human somatic cell hybrids with an apo(a) cDNA probe revealed a complex pattern of bands, all of which segregated with chromosome 6. In situ hybridization yielded a single peak of grain density located on chromosome 6q26–27. Apo(a) cDNA sequences exhibit striking homology to those of the plasma protease plasminogen, and, therefore, we have reexamined the chromosome assignment of the plasminogen gene. We conclude that both the apo(a) and plasminogen genes reside on human chromosome 6q22–27, consistent with a gene duplication mechanism for their evolutionary origin. The results are of significance for the genetic control of apo(a) expression and genetic influences predisposing to atherosclerosis.     

Genetic linkage between lipoprotein(a) phenotype and a DNA polymorphism in the plasminogen gene

Coronary heart disease risk correlates directly with plasma concentrations of lipoprotein(a) (Lp(a)), a low-density lipoprotein-like particle distinguished by the presence of the glycoprotein apolipoprotein(a) (apo(a)), which is bound to apolipoprotein B-100 (apoB-100) by disulfide bridges. Size isoforms of apo(a) are inherited as Mendelian codominant traits and are associated with variations in the plasma concentration of lipoprotein(a). Plasminogen and apo(a) show striking protein sequence homology, and their genes both map to chromosome 6q26-27. In a large family with early coronary heart disease and high plasma concentrations of Lp(a), we found tight linkage between apo(a) size isoforms and a DNA polymorphism in the plasminogen gene; plasma concentrations of Lp(a) also appeared to be related to genetic variation at the apo(a) locus. We found free recombination between the same phenotype and alleles of the apoB DNA polymorphism. This suggests that apo(a) size isoforms and plasma lipoprotein(a) concentrations are each determined by genetic variation at the apo(a) locus.     

Two copies of the human apolipoprotein C-I gene are linked closely to the apolipoprotein E gene

The gene for human apolipoprotein (apo) C-I was selected from human genomic cosmid and lambda libraries. Restriction endonuclease analysis showed that the gene for apoC-I is located 5.5 kilobases downstream of the gene for apoE. A copy of the apoC-I gene, apoC-I', is located 7.5 kilobases downstream of the apoC-I gene. Both genes contain four exons and three introns; the apoC-I gene is 4653 base pairs long, the apoC-I' gene 4387 base pairs. In each gene, the first intron is located 20 nucleotides upstream from the translation start signal; the second intron, within the codon of Gly-7 of the signal peptide region; and the third intron, within the codon for Arg39 of the mature plasma protein coding region. The upstream apoC-I gene encodes the known apoC-I plasma protein and differs from the downstream apoC-I' gene in about 9% of the exon nucleotide positions. The most important difference between the exons results in a change in the codon for Gln-2 of the signal peptide region, which introduces a translation stop signal in the downstream gene. Major sequence differences are found in the second and third introns of the apoC-I and apoC-I' genes, which contain 9 and 7.5 copies, respectively, of Alu family sequences. The apoC-I gene is expressed primarily in the liver, and it is activated when monocytes differentiate into macrophages. In contrast, no mRNA product of the apoC-I' gene can be detected in any tissue, suggesting that it may be a pseudogene. The similar structures and the proximity of the apoE and apoC-I genes suggest that they are derived from a common ancestor. Furthermore, they may be considered to be constituents of a family of seven apolipoprotein genes (apoE, -C-I, -C-II, -C-III, -A-I, -A-II, and -A-IV) that have a common evolutionary origin.     

The apolipoprotein (a) gene: a transcribed hypervariable locus controlling plasma lipoprotein (a) concentration

Lipoprotein(a) [Lp(a)] is a quantitative trait in human plasma. Lp(a) consists of a low-density lipoprotein and the plasminogen-related apolipoprotein(a) [apo(a)]. The apo(a) gene determines a size polymorphism of the protein, which is related to Lp(a) levels in plasma. In an attempt to gain a deeper insight into the genetic architecture of this risk factor for coronary heart disease, we have investigated the basis of the apo(a) size polymorphism by pulsed field gel electrophoresis of genomic DNA employing various restriction enzymes (SwaI, KpnI, KspI, SfiI, NotI) and an apo(a) kringle-IV-specific probe. All enzymes detected the same size polymorphism in the kringle IV repeat domain of apo(a). With KpnI, 26 different alleles were identified among 156 unrelated subjects; these alleles ranged in size from 32kb to 189kb and differed by increments of 5.6kb, corresponding to one kringle IV unit. There was a perfect match between the size of the apo(a) DNA phenotypes and the size of apo(a) isoforms in plasma. The apo(a) DNA polymorphism was further used to estimate the magnitude of the apo(a) gene effect on Lp(a) levels by a sib-pair comparison approach based on 253 sib-pairs from 64 families. Intra-class correlation of log-transformed Lp(a) levels was high in sib-pairs sharing both parental alleles (r = 0.91), significant in those with one common allele (r = 0.31), and absent in those with no parental allele in common (r = 0.12). The data show that the intra-individual variability in Lp(a) levels is almost entirely explained by variation at the apo(a) locus but that only a fraction (46%) is explained by the DNA size polymorphism. This suggests further heterogeneity relating to Lp(a) levels in the apo(a) gene.     

Familial apolipoprotein CII deficiency: A preliminary analysis of the gene defect in two independent families

Summary We have used a cDNA clone for human apolipoprotein CII (apo CII) to study the apo CII genes in two independent individuals with familial apo CII deficiency. With all the restriction enzymes so far used, gene fragments hybridising with apo CII cDNA are observed that are indistinguishable from normal samples. This demonstrates that in neither of these individuals is the defect due to a major deletion of DNA in or around the apo CII gene. We have used a common polymorphism of the apo CII gene detected with the enzyme TaqI to follow the inheritance of the gene in the families of these apo CII deficient individuals. The pattern of inheritance that we observe is consistent with the defect causing apo CII deficiency being in, or closely linked to the apo CII structural gene.     

Structure and expression of the gene coding for the human serpin hLS2

We have analyzed genomic clones encoding human leuserpin 2 (hLS2). The gene covers about 14.5 kilobases and consists of 5 exons and 4 introns. The genes coding for hLS2, alpha 1-antitrypsin, alpha 1-antichymotrypsin, and rat angiotensinogen share an equivalent exon-intron structure and therefore constitute a distinct subgroup within the serpin gene family, which otherwise displays a highly variable exon-intron pattern. With the exception of a segment in the second exon, the sequence similarity of the genes coding for hLS2 and alpha 1-antitrypsin extends to all exons including one encoding the 5'-untranslated sequences. The implications of these findings with respect to the genesis of the amino-terminal heterogeneity in the serpin family are discussed.     

A 10-bp deletion in the apolipoprotein epsilon gene causing apolipoprotein E deficiency and severe type III hyperlipoproteinemia.

Type III hyperlipoproteinemia (HLP) is usually associated with homozygosity for apolipoprotein (apo) E2. We identified a 30-year-old male German of Hungarian ancestry with severe type III HLP and apo E deficiency. The disease was expressed in an extreme phenotype with multiple cutaneous xanthomas. Apo E was detectable only in trace amounts in plasma but not in the different lipoprotein fractions. Direct sequencing of PCR-amplified segments of the apo epsilon gene identified a 10-bp deletion in exon 4 (bp 4037-4046 coding for amino acids 209-212 of the mature protein). The mutation is predictive for a reading frameshift introducing a premature stop codon (TGA) at amino acid 229. By western blot analysis, we found small amounts of a truncated apo E in the patient's plasma. Family analysis revealed that the proband was homozygous--and 10 of 24 relatives were heterozygous--for the mutation. Heterozygotes had, as compared to unaffected family members, significantly higher triglycerides (TG), very low-density lipoprotein (VLDL) cholesterol and a significantly higher VLDL cholesterol-to-serum TG ratio, which is indicative of a delayed remnant catabolism. We propose that the absence of a functionally active apo E is the cause of the severe type III HLP in the patient and that the mutation, even in a single dose in heterozygotes, predisposes in variable severity to the phenotypic expression of the disease.     

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.     

The Peculiar Evolution of Apolipoprotein(a) in Human and Rhesus Macaque

Apo(a) is a low density lipoprotein homologous to plasminogen and has been shown to be involved in coronary atheroschlerosis. In the present paper we will try to analyze the interesting evolutionary pattern of Apo(a). The plasminogen gene contains 5 cysteine-rich sequences, called kringles, followed by a protease domain. Apo(a), probably arisen by duplication of an ancestral plasminogen gene, contains many tandemly repeated copies of a sequence domain similar to the fourth kringle of plasminogen, 37 in human and at least 10 in the partially sequenced gene of rhesus, and the protease domain. We have found that the upstream kringles of apo(a) undergo Molecular Drive-like processes that produce high intraspecies similarity, whereas the downstream kringles evolve in a molecular clock-like manner and show an high interspecies sequence similarity. The latter regions are obviously suitable for dating the duplication event by which Apo(a) arose from plasminogen, but only if they evolve at the same rate in the two genes. Thus, we propose a ``Molecular Clock Test' for assessing whether the comparison of two paralogous genes (or gene regions) can give reliable information on the dating of their origin by duplication. Applying this test to the kringle-4 domain of apo(a) and plasminogen gene, we demonstrate that the separation between the two genes by duplication dates back at about 90 Mya immediately before the radiation of mammals.     

Structure, organization, and chromosomal location of the gene encoding a form of rice soluble starch synthase.

A rice (Oryza sativa L.) genomic clone encoding the gene for a form of soluble starch synthase (SSS1) and its 5'- and 3'-flanking regions has been isolated and sequenced. The SSS1 gene contained 15 exons interrupted by 14 introns. The exon/intron organization of the SSS1 gene was divergent from that of the rice Waxy gene coding for granule-bound starch synthase, thus suggesting that the SSS1 and granule-bound starch synthase genes have evolved from an ancestral gene in a different way or that the two genes are products of different ancestral genes that have converged during evolution. However, these two genes were closely located to each other on rice chromosome 6 at an approximate map distance of 5 centimorgans. The nucleotide sequence of the 5'-end region of the gene is unique because of the presence of some repetitive sequences.     

Evolution of the EF-hand calcium-binding protein family: Evidence for exon shuffling and intron insertion

Summary The evolutionary history of the intracellular calcium-binding protein superfamily is well documented. The members of this gene family are all believed to be derived from a common ancestor, which, itself, was the product of two successive gene duplications. In this study, we have compared and analyzed the structures of the recently described genes coding for these proteins. We propose a series of evolutionary events, which include exon shuffling and intron insertion, that could account for the evolutionary origin of all the members of this super-family. According to this hypothesis, the ancestral gene, a product of two successive duplications, consisted of at least four exons. Each exon coding for a peptide (a calcium-binding domain) was separated by an intron that had mediated the duplication. Each distinct lineage evolved from this ancestor by genomic rearrangement, with insertion of introns being a prominent feature.     

Evolution of the apolipoproteins. Structure of the rat apo-A-IV gene and its relationship to the human genes for apo-A-I, C-III, and E

We have determined the nucleotide sequence of the rat apolipoprotein (apo-) A-IV gene and analyzed its structural and evolutionary relationships to the human apolipoprotein A-I, E, and C-III genes. The rat A-IV gene is 2.4 kilobases in size and consists of three exons (142, 126, and 1157 base pairs) interrupted by two introns (277 and 673 base pairs). The 5'-nontranslated region and most of the signal peptide are encoded by the first exon. Thus, the apo-A-IV gene lacks an intron in the 5'-nontranslated region of its mRNA in contrast to all other known apolipoprotein genes. Sequences coding for amphipathic docosapeptides span both the second and third exons of the rat A-IV gene. We demonstrate that this is also true for the human apolipoprotein genes. This gene family seems to have evolved by the duplication of an ancestral minigene that resulted in the formation of two exons. Thereafter, evolution of these sequences was dominated by intraexonic amplification of repeating units coding for amphipathic peptides. Sequence divergence of these repeats resulted in the functional differentiation of the apolipoproteins. However, conservation of the fundamental amphipathic pattern allowed members of this protein family to retain their lipid-binding properties.     

Studies on the organization of the human apolipoprotein B 100 gene

The organization of five exons of the 3' terminal end of the human apolipoprotein B 100 (apo B 100) gene 1906, 184, 115, 7572 and 374 bp long have been determined from two overlapping EMBL3 human genomic clones extending over 18 kb. They encode more than 70% of the apo B 100 amino-acid sequence. The introns between these five exons were sequenced revealing the common intron/exon splice junction sequences. The 7572 bp exon is the longest exon so far reported for mammalian genes with the proposed sequence coding for the LDL receptor binding site. Its possible relationship to apolipoprotein B 48 is discussed.