Characterization of the human platelet glycoprotein IIIa gene. Comparison with the fibronectin receptor beta-subunit gene

This study was designed to determine the structure of the gene for glycoprotein (GP) GPIIIa, the beta-subunit of the platelet membrane GPIIb-IIIa complex. The complexity of the gene was determined after Southern analysis of human chromosomal DNA. Overlapping genomic clones were isolated from cosmid and phage lambda libraries that contained the entire coding unit of the human gene for the mature GPIIIa protein. The genomic clones spanned approximately 60 kilobase pairs of human DNA sequence. The exon containing segments of the clones was mapped and the exons, including the exonintron junctions, were sequenced. The GPIIIa protein is divided into 14 exons ranging in size from 87 to 430 nucleotides separated by introns, which were 0.3 to 9 kilobase pairs in size. The 3' exon was larger than 1700 nucleotides and contained the 3'-untranslated region. Several structural domains of the GPIIIa protein were contained within individual exons. These included (i) the transmembrane spanning segment, (ii) the cytoplasmic region containing the potential phosphorylation sites, and (iii) the six domains in the NH2-terminal half of GPIIIa that are highly conserved between two other integrin beta-subunits. In contrast, other domains such as the four cysteine-rich repeats were interrupted by introns. Genomic clones for the beta-subunit of the fibronectin receptor (beta 1) were also isolated, partially mapped, and sequenced. Of the eight splice sites identified in beta 1, six occurred at the same amino acid residue in GPIIIa. These results provide genetic evidence that GPIIIa and beta 1 have a common evolutionary origin within the integrin family.     

Partial structure of the gene for chicken cartilage proteoglycan core protein

A genomic DNA fragment (gCORE-1), encoding a portion of the cartilage proteoglycan core protein, has been isolated from a phage library using cDNA as a probe. The genomic insert is about 17 kilobase pairs; two BamHI fragments of the insert (1.3 and 4.8 kilobase pairs) contain most of the hybridizable sequences found in the cDNA. Sequence analysis of these fragments shows that they contain a total of five exons that encompass 216 amino acid residues, all of which are identical to those of the corresponding cDNA sequence. Three of the exons, which are adjacent to one another, are very similar to the corresponding exons in the gene of a rat hepatic lectin as well as to an exon in the gene of human pulmonary surfactant-associated protein. There is a strong degree of conservation of amino acid sequences encoded in the three genes, although there is no similarity between their introns. The sizes of the five exons in gCORE-1, except for one (which is indeterminate because only a partial cDNA sequence is available), are less than 184 base pairs, whereas the sizes of the introns range from 218 to greater than 2629 base pairs. Four of the introns interrupt an exon codon at either their donor or acceptor sites, between the first and second nucleotides. Only one intron does not split a codon. Intron and exon boundary sites are in agreement with known consensus sequences for introns. The dispersed distribution and relatively small size of the exons, if representative of the entire gene, suggest that the complete gene which codes for the core protein may be quite sizable.     

Structure of the human type I DNA topoisomerase gene

We describe the molecular organization of the human gene coding for type I DNA topoisomerase. The coding sequence is split into 21 exons distributed over at least 85 kilobase pairs (kb) of human genomic DNA. The sizes of the 20 introns vary widely between 0.2 and at least 30 kb and all contain the sequence elements known to be required for pre-mRNA splicing. Several of the intron sequences separate exons encoding parts of the enzyme that are highly conserved between human and yeast suggesting that at least some of the exons may code for individual, structurally, or functionally important domains of the enzyme. We also describe the promoter sequence of the human topoisomerase I gene and show that it is composed of distinct functional elements.     

Characterization of the bovine prothrombin gene

The bovine prothrombin gene was characterized by Southern blot analysis of bovine genomic DNA using bovine prothrombin cDNA fragments as hybridization probes. These analyses suggested that the bovine genome contains a single prothrombin gene that is at least 10 kilobase pairs (kbp) in size. To characterize the gene more thoroughly, two bovine genomic phage libraries were screened by using prothrombin cDNAs as hybridization probes. Heteroduplex analysis of the cloned genomic DNA and cDNA showed that the prothrombin gene is 14.9 kbp in size and contains at least 14 exons interrupted by 13 introns. The exons vary in size from 28 to 317 base pairs (bp), while the introns vary in size from less than 100 to 6940 bp. Regions of self-complementarity were observed within some of the introns, suggesting the presence of inverted repeat sequences. The bovine prothrombin gene shows similarities in structure to both the human prothrombin gene and the human factor IX gene.     

Genomic structure of the human mitochondrial aldehyde dehydrogenase gene

We have isolated and characterized four overlapping clones from two cosmid human genomic libraries, which span about 90 kilobase pairs (kbp) and contain the entire human mitochondrial aldehyde dehydrogenase (ALDH2) gene. Restriction maps of the genomic clones were elucidated utilizing cDNA probes and specific oligonucleotide probes. The organization of exons and introns was established by DNA sequencing of each exon and splicing junctions. The ALDH2 gene is about 44 kbp in length and contains at least 13 exons which encode 517 amino acid residues. Except for the signal NH2-terminal peptide, which is absent in the mature enzyme, the amino acid sequence deduced from the exons coincided with the reported primary structure of human liver ALDH2 (J. Hempel, R. Kaiser, and H. J?rnvall, 1985, Eur. J. Biochem. 153: 13-28). Several introns contain Alu repetitive sequences. A TATA-like sequence (TTATAAAA) and a CAAT-like sequence (GTCATCAT) are located 473 and 515 bp, respectively, upstream from the translation initiation codon. Primer extension and S1 nuclease mapping were performed to characterize the 5'-region of the gene.     

Nucleotide sequence of the gene for the b subunit of human factor XIII

Factor XIII (Mr 320,000) is a blood coagulation factor that stabilizes and strengthens the fibrin clot. It circulates in blood as a tetramer composed of two a subunits (Mr 75,000 each) and two b subunits (Mr 80,000 each). The b subunit consists of 641 amino acids and includes 10 tandem repeats of 60 amino acids known as GP-I structures, short consensus repeats (SCR), or sushi domains. In the present study, the human gene for the b subunit has been isolated from three different genomic libraries prepared in lambda phage. Fifteen independent phage with inserts coding for the entire gene were isolated and characterized by restriction mapping, Southern blotting, and DNA sequencing. The gene was found to be 28 kilobases in length and consisted of 12 exons (I-XII) separated by 11 intervening sequences. The leader sequence was encoded by exon I, while the carbonyl-terminal region of the protein was encoded by exon XII. Exons II-XI each coded for a single sushi domain, suggesting that the gene evolved through exon shuffling and duplication. The 12 exons in the gene ranged in size from 64 to 222 base pairs, while the introns ranged in size from 87 to 9970 nucleotides and made up 92% of the gene. The introns contained four Alu repetitive sequences, one each in introns A, E, I, and J. A fifth Alu repeat was present in the flanking 3' end of the gene. Two partial KpnI repeats were also found in the introns, including one in intron I and one in intron J. The KpnI repeat in intron J was 89% homologous to a sequence of approximately 2200 nucleotides flanking the gene coding for human beta globin and approximately 3800 nucleotides from the L1 insertion present in the gene for human factor VIII. Intron H also contained an "O" family repeat, while two potential regions for Z-DNA were identified within introns G and J. One nucleotide change was found in the coding region of the gene when its sequence was compared to that of the cDNA. This difference, however, did not result in a change in the amino acid sequence of the protein.     

Organization of the human hepatocyte growth factor-encoding gene.

Human genomic phage libraries were screened for the human hepatocyte growth factor (HGF)-encoding gene (HGF) using a cDNA encoding the human protein as a probe. Characterization of the clones revealed that this gene is composed of 18 exons interrupted by 17 introns spanning approx. 70 kb. The first exon contains the 5'-untranslated region and the signal peptide. The next ten exons encode the alpha-chain which contains four kringle structures. Each kringle domain is encoded by two exons as observed in other kringle-containing proteins. The twelfth exon contains the short spacer region between the alpha- and beta-chains and the remaining six exons comprise the beta-chain. The beta-chain is structurally similar to the catalytic domains of serine proteases; amino acid substitutions in the active site were found. The organization of the HGF gene is highly homologous to those of the serine proteases involved in blood coagulation and fibrinolysis, especially with that of plasminogen. This suggests that the human HGF gene is evolutionally related to these genes.     

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.     

Structure of the human prealbumin gene

Using cloned human prealbumin cDNA as a probe, Southern blot hybridization of human genomic DNA revealed that the prealbumin gene consists of an unique, single-copy DNA. The nucleotide sequences of the entire human prealbumin gene, including both 581 base pairs of the 5'- and 95 base pairs of the 3'-flanking sequences, were determined. The gene spans about 7.0 kilobase pairs and consists of four exons and three introns. As in most eukaryotic genes, the consensus TATA and CAAT sequences are found 30 and 101 nucleotides, respectively, upstream from the putative cap site, and a polyadenylation signal sequence AA-TAAA is found in the 3'-untranslated region. Unexpectedly, two independent open reading frames provided with respective regulatory sequences were found within the gene: one in the first intron and the other in the third intron.     

Structure of the human laminin B2 chain gene reveals extensive divergence from the laminin B1 chain gene

The exon-intron structure of the human laminin B2 chain gene was elucidated from genomic lambda phage clones spanning 2 kilobase pairs (kb) of the 5'-flanking region, 58 kb of the structural gene and 10 kb of the 3'-flanking region. The entire gene was shown to contain 28 exons. The promoter region has no TATA or CAAT boxes whereas it contains five GC boxes and three AP-2-like binding sites. Comparison with the promoter region of the mouse gene revealed six highly conserved sequences of 14 to 42 base pairs in length. Sequencing of the last exon of the gene showed that the 3'-untranslated region of the mRNA can be up to 2797 nucleotides with five AATAAA potential polyadenylation signals. The similarity of the human 3'-untranslated sequence with that of mouse was shown to be 68.8%. The exon-intron structure of the laminin B2 chain gene demonstrated extensive divergence from the human laminin B1 chain gene, which has 34 exons. Only three intron locations are conserved in these two genes. The overall exon profile of the laminin B2 chain gene correlates only marginally with the pattern of structural domains and internal cysteine-rich repeats in the laminin B2 polypeptide chain.     

Characterization of the human cyclophilin gene and of related processed pseudogenes

The human cyclophilin gene was isolated from a genomic library derived from leucocyte DNA and sequenced. The gene contains five exons and four introns. The amino acid sequence deduced from the exons matches perfectly the one previously determined from the T-cell cyclophilin cDNA. A TATA box is visible in the promoter region and putative Sp1 binding sites are also found there as well as in the first intron. Six members of the middle repetitive Alu gene family are present in one or other orientation in the non-coding regions of the cyclophilin gene. Hybridisation of genomic DNA to probes derived from the promoter region or the first intron indicates that the cyclophilin gene is present as a single copy in the human haploid genome. Seven other cyclophilin-related DNA clones isolated from the same library were also characterized. They show a high degree of similarity to the cyclophilin cDNA and are colinear to it. However, multiple genetic lesions, often including deletion and/or insertion events which modify the reading frame, are found in these clones which are therefore likely to represent processed pseudogenes.     

Structural organization of the rat gene for the arginine vasopressin-neurophysin precursor.

The rat arginine vasopressin-neurophysin precursor gene has been isolated from a genomic library cloned in lambda phage Charon 4A. Restriction mapping and nucleotide sequence analysis demonstrated that the gene is 1.85 kilobase pairs long and contains two intervening sequences located in the protein coding region. Exon A encodes a putative signal peptide, the hormone arginine vasopressin and the variable N terminus of the carrier protein neurophysin, exon B encodes the highly conserved middle part of neurophysin and exon C its variable C terminus together with glycoprotein. Thus, the three functional domains of the percursor - arginine, vasopressin, neurophysin, glycoprotein - are encoded on three distinct exons.