Characterization of the gene encoding the major rat liver asialoglycoprotein receptor

A cloned cDNA encoding the major rat liver asialoglycoprotein receptor has been used to analyze the gene for this protein. Genomic Southern blot analysis reveals that the gene is contained on a single EcoRI restriction fragment and is unique. A clone containing the gene (isolated from a rat liver genomic library) has been characterized by sequence analysis. The mRNA for the receptor is encoded by nine exons separated by eight introns. The first exon is confined to the 5'-untranslated region of the mRNA, the second exon encodes most of the cytoplasmic NH2-terminal domain of the receptor polypeptide, the third exon corresponds to the hydrophobic transmembrane portion of the polypeptide, and the remaining exons encode the extracellular parts of the receptor. Some, but not all, of the divisions between exons correspond to boundaries between functional domains of the polypeptide.     

Evolutionary conservation of intron position in a subfamily of genes encoding carbohydrate-recognition domains

The structure of the gene encoding a chicken liver receptor, the chicken hepatic lectin, which mediates endocytosis of glycoproteins has been established. The coding sequence is divided into six exons separated by five introns. The first three exons correspond to separate functional domains of the receptor polypeptide (cytoplasmic tail, transmembrane sequence, and extracellular neck region), while the final three exons encode the Ca(2+)-dependent carbohydrate-recognition domain. These results, as well as computer-assisted multiple sequence comparisons, establish this receptor as the evolutionary homolog of the mammalian asialoglycoprotein receptors. It is interesting that the chicken receptor falls into a subfamily of proteins along with the mammalian asialoglycoprotein receptors, since the saccharide-binding specificity of the chicken receptor resembles more closely that of a different set of calcium-dependent animal lectins, which includes the mannose-binding proteins. The portions of the genes encoding the carbohydrate-recognition domains of these proteins lack introns. The results suggest that divergence of intron-containing and intron-lacking carbohydrate-recognition domains preceded shuffling events in which other functional domains were associated with the carbohydrate-recognition domains. This was followed by further divergence, generating a variety of saccharide-binding specificities.     

Chromosomal Location and Some Structural Features of Human Clathrin Light-Chain Genes (CLTA and CLTB)

Two human clathrin light-chain genes have been defined. The gene (CLTA) encoding the LCa light chain maps to the long arm of chromosome 12 at 12q23-q24 and that encoding the LCb light chain (CLTB) maps to the long arm of chromosome 4 at 4q2-q3. Isolation and characterization of partial genomic clones encoding human LCa and LCb reveal the neuron-specific insertions of the LCa and LCb proteins to he encoded by discrete exons, thus proving that clathrin light chains undergo alternate mRNA splicing to generate tissue-specific protein isoforms. The insertion sequence of LCb is encoded by a single exon and that of LCa by two exons. The first of the two neuron-specific LCa exons is homologous to the corresponding LCb exon. An intronic sequence of the LCb gene with similarity to the second neuron-specific exon of the LCa gene has been identified.     

Mannose-binding proteins isolated from rat liver contain carbohydrate-recognition domains linked to collagenous tails. Complete primary structures and homology with pulmonary surfactant apoprotein

Preparations of mannose-binding protein isolated from rat liver contain two distinct but homologous polypeptides. The complete primary structures of both of these polypeptides have been determined by sequencing of peptides derived from the proteins, isolation and sequencing of cDNAs for both proteins, and partial characterization of the gene for one of the proteins. Each polypeptide consists of three regions: (a) an NH2-terminal segment of 18-19 amino acids which is rich in cysteine and appears to be involved in the formation of interchain disulfide bonds which stabilize dimeric and trimeric forms of the protein, (b) a collagen-like domain consisting of 18-20 repeats of the sequence Gly-X-Y and containing 4-hydroxyproline residues in several of the Y positions, and (c) a COOH-terminal carbohydrate-binding domain of 148-150 amino acids. The sequences of the COOH-terminal domains are highly homologous to the sequence of the COOH-terminal carbohydrate-recognition portion of the chicken liver receptor for N-acetylglucosamine-terminated glycoproteins and the rat liver asialoglycoprotein receptor. Each protein is preceded by a cleaved, NH2-terminal signal sequence, consistent with the finding that this protein is found in serum as well as in the liver. The entire structure of the mannose-binding proteins is homologous to dog pulmonary surfactant apoprotein.     

Isolation and sequence of a rat chymotrypsin B gene

A cDNA clone encoding part of chymotrypsin B was isolated from a cDNA library prepared from rat pancreatic mRNA and used as a probe to isolate the chymotrypsin B gene. The nucleotide sequence of this gene is presented. The 4709-base pair transcribed portion of the isolated gene was inferred from the cDNA and gene sequence, and the 5' border was determined by primer extension on pancreatic polyadenylated RNA. The coding portion of the gene is interrupted by six introns. The active site residues His 57, Asp 102, and Ser 195 are encoded by separate exons. Moreover, two regions of the enzyme which form the substrate-binding pocket are also encoded by separate exons. Thus, the substrate specificity and catalytic activity of the enzyme are produced by joining several exons encoding protein segments that are intrinsically catalytically inactive. The number and location of the intron/exon junctions of the chymotrypsin gene as compared to those of other serine protease genes, as well as the location of the genes on separate chromosomes, suggest that the duplication that resulted in the formation of the chymotrypsin gene was an ancient evolutionary event.     

Characterization of two mannose-binding protein cDNAs from rhesus monkey (Macaca mulatta): structure and evolutionary implications

Mannose-binding proteins (MBPs), members of the collectin family,have been implicated as lectin opsonins for various virusesand bacteria. Two distinct but related MBPs, MBP-A and MBP-C,with -55% identity at the amino acid level, have been previouslycharacterized from rodents. In humans, however, only one formof MBP has been characterized. In this paper we report studieselucidating the evolution of primate MBPs. ELISA and Westernblot analyses indicated that rhesus and cynomolgus monkeys havetwo forms of MBP in their sera, while chimpanzees have onlyone form, similar to humans. Two distinct MBP cDNA clones wereisolated and characterized from a rhesus monkey liver cDNA library.Rhesus MBP-A is closely related to the mouse and rat MBP-A,showing 77% and 75% identity at the amino acid level, respectively.Rhesus MBP-A also has three cysteines at the N-terminus, similarto mouse and rat MBP-A and human MBP. Rhesus MBP-C shares 90%identity with the human MBP at the amino acid level and hasthree cysteines at the N-terminus, in contrast to two cysteineresidues found in rodent MBP-C. A stretch of nine amino acidsclose to the N-terminus, absent in both mouse and rat MBP-A,but present in rodent MBP-C, chicken and human MBPs, is alsofound in the rhesus MBP-A. The phylogenetic analysis of rhesusand other mammalian MBPs, coupled with the serological datasuggest that at least two distinct MBP genes existed prior tomammalian radiation and the hominoid ancestor apparently lostone of these genes or failed to express it. collectin rhesus monkey mannose-binding protein MBP cDNA mannan-binding protein     

The Human Lamin B Receptor/Sterol Reductase Multigene Family

LBR (lamin B receptor) is an integral protein of the inner nuclear membrane encoded by a gene on human chromosome 1q42.1. LBR has a nucleoplasmic, amino-terminal domain of approximately 200 amino acids followed by a carboxyl-terminal domain similar in sequence to yeast and plant sterol reductases. We have determined the primary structures of two human proteins with strong sequence similarity to the carboxyl-terminal domain of LBR and sterol reductases. Their genes have recently been assigned the symbols TM7SF2 and DHCR7. TM7SF2 mRNA is most predominantly expressed in heart and DHCR7 mRNA mostly in liver and brain. Whereas LBR is localized to the inner nuclear membrane, these two related proteins are in the endoplasmic reticulum. TheTM7SF2gene contains 10 coding exons, and its intron positions are exactly conserved in the part of theLBRgene encoding its carboxyl-terminal domain. Intron positions in theDHCR7gene are also similar. Both of these new LBR-like genes are on chromosome 11q13. These results describe a human gene family encoding proteins of the inner nuclear membrane and endoplasmic reticulum that function in nuclear organization and/or sterol metabolism.     

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.     

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.     

Evolution of aspartyl proteases by gene duplication: the mouse renin gene is organized in two homologous clusters of four exons.

Overlapping recombinant clones that appear to encompass the entire renin gene, named Ren 1, have been isolated from a library of BALB/c mouse genomic DNA fragments. Based on restriction endonuclease mapping and DNA sequence analysis, Ren 1 spans 9.6 kb and contains nine exons interrupted by eight intervening sequences of highly variable size. The first exon, encoding the signal peptide of preprorenin, is separated from the eight following exons by a 3-kb intron. These eight exons are organized into two clusters of four separated by a 2-kb intron. DNA stretches encoding the aspartyl residues, which are part of the active site of renin, are located at homologous positions in both clusters. Our results show that aspartyl protease genes have arisen by duplication and fusion of an ancestral gene containing five exons. The estimated date of the duplication event of the mouse renin genes Ren 1 and Ren 2 is discussed.     

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.     

Alpha-tropomyosin gene organization. Alternative splicing of duplicated isotype-specific exons accounts for the production of smooth and striated muscle isoforms

We have previously isolated and characterized cloned complementary DNAs (cDNAs) for striated and smooth muscle alpha-tropomyosin. The sequences of these cDNA clones suggested that these two isoforms were encoded by the same gene. Here, we have determined the complete structure of the alpha-tropomyosin (alpha-TM) gene, establishing that a single gene, with a sequence complexity of 28 kilobase pairs, is split into 12 exons and produces the smooth and striated muscle alpha-TM mRNA isoforms by alternative splicing of a minimum of five exchangeable isotype-specific exons. The elucidation of the intron/exon organization of alpha-TM suggests that this gene evolved from an ancestral gene encoding a 21-aa protein that might represent the primordial actin binding domain. Sequence comparison between the pairs of exons coding for the "isotype switch regions" and among the corresponding regions of tropomyosin genes in a variety of species ranging from insects to mammals, suggests that the alternatively spliced exons are very old and might have arisen before the radiation of the arthropods, more than 600 million years ago. Additionally, the examination of the intronic sequences has uncovered potential alternative intramolecular secondary structures (hairpin-loop structures) which might be involved in the tissue-specific expression of the duplicated and mutually exclusive alpha-TM isotype-specific exons.     

Structure, chromosomal localization and evolutionary conservation of the gene encoding human U1 snRNP-specific A protein.

Three specific proteins, called A, 70K and C, are present in the U1 small nuclear ribonucleoprotein (snRNP) particle, in addition to the common proteins. The human U1 snRNP-specific A protein is, apart from a proline-rich region, highly similar to the U2 snRNP-specific protein B". To examine the homologous regions at the genomic level, we isolated and characterized the human U1-A gene. The human U1-A protein appears to be encoded by a single-copy gene and its locus has been mapped to the q arm of chromosome 19. The gene, about 14-16 kb in length, consists of six exons. The regions homologous to the U2-B" gene are not limited to single exons and are mostly not confined by exon-exon junctions in the corresponding U1-A mRNA. However, the proline-rich region of U1-A, absent in U2-B", is encoded by a single exon, suggesting a specific function for this domain of U1-A. The region of the cap site and upstream sequences contain interesting similarities to the promoter region of other snRNP protein-encoding genes and several housekeeping genes, in particular the vertebrate ribosomal protein-encoding genes. Hybridization experiments with various vertebrate genomic DNAs revealed that U1-A sequences are evolutionarily conserved in all tested vertebrate genomes, except for chicken, duck and pigeon. The divergence of these avian genomes is probably typical for the class of birds.     

Piscine glycoprotein hormone (gonadotropin and thyrotropin) receptors: a review of recent developments

Similar to the higher vertebrates, the pituitary in bony fishes express three glycoprotein hormones: thyroid-stimulating hormone (TSH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH). In addition to the appropriate secretion of these hormones, the timely and quantitative expression of their specific receptors (TSHR, FSHR and LHR) in the target tissues is an essential requirement for their physiological action. In fishes that constitute a very diverse group of vertebrates, there are only a few published reports of primary structure of these receptors although other examples have been communicated briefly. This review will summarize these reports as well as to describe the insights gained from what is known about the mammalian receptors. The structural organization of the fish receptors (as deduced from the encoding cDNAs) is highly homologous to the higher vertebrate receptors in that there is a 7-pass transmembrane region and an N-terminal extracellular domain, which contributes to ligand specificity. In mammals, the FSHR and the TSHR genes are composed of 10 exons whereas the LHR gene is composed of 11 exons. The position of the 'extra intron' is conserved in the catfish LHR gene. In the mammals, the transmembrane domain of each of the three glycoprotein hormone receptors is encoded by a single exon, however, in the salmon genes and homologous invertebrate genes, this portion of the receptor is encoded by multiple exons. In general, the tissue-specific expression of these receptors is similar to that seen in mammals, however, the gonadal expression of TSHR in the striped bass and sunrise sculpin and the renal expression of LHR in the channel catfish are unique.