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, sequence, and polymorphism of the Lyt-2 T cell differentiation antigen gene

We have determined the nucleotide sequence and genomic organization of the mouse Lyt-2 T lymphocyte differentiation antigen gene. This gene consists of five exons and four introns, and the organization roughly parallels the protein domains. Alternative splicing to include or exclude exon IV (encoding part of the cytoplasmic tail) results in two forms of mRNA and accounts for the difference in size between the alpha- and alpha'-chains of Lyt-2. The gene structure provides further evidence for the evolutionary relationship between Lyt-2 and immunoglobulin genes. Comparison of the nucleotide sequence of the Lyt-2.1 and Lyt-2.2 alleles shows a high degree of conservation, but indicates that a single nucleotide change and consequent amino acid substitution in the variable region-like domain accounts for the serologic difference between these two alleles.     

Structure of the murine mb-1 gene encoding a putative sIgM-associated molecule

Genomic DNA clones containing the B cell-specific murine mb-1 gene were isolated and a 5.6-kb BamH I fragment was characterized. It is 5629 bp long and contains five exons: an exon containing the 5' untranslated and the coding sequence of the signal peptide, an exon of 294 bp, which contains most of the extracellular sequence of the MB-1 protein, a 119-bp long exon coding mainly for the transmembrane portion, and two exons of 69 bp and 427 bp encoding the cytoplasmic domain and the 3'-untranslated region, respectively. The mb-1 gene does not contain a "TATA box" found in many eukaryotic promoters. The 5'-flanking region has sequence stretches homologous to IgVH 5'-promoter regions and a bcl 2 intron sequence. It contains the decanucleotide sequence (ATGGCAAATA) almost identical to the octamer motif of IgVH promoters. A B cell-specific DNase I-hypersensitive site was found in the 3'-flanking region indicating that this region might be involved in B cell-specific expression of mb-1. Southern blot analysis of genomic liver DNA with the cloned mb-1 cDNA suggests the existence of another mb-1-related gene segment.     

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.     

Genomic cloning of the gene for an IgE-binding lectin reveals unusual utilization of 5' untranslated regions.

epsilon BP (for epsilon binding protein) is a M(r) 31,000 S-type animal lectin that binds to IgE and has been identified as the homologue of Mac-2, a macrophage cell-surface marker, as well as the lectins RL-29, CBP35, and L-34. The protein is composed of two domains with the amino-terminal portion containing tandem repeats of nine amino acids and the carboxyl-terminal half containing consensus sequences shared by S-type animal lectins. We determined the genomic map in both rat and mouse and isolated overlapping genomic clones that contain the 5' two-thirds of the murine gene. The remaining portion of the gene was obtained by polymerase chain reaction (PCR) amplification of genomic murine DNA followed by subcloning into plasmid vectors. The epsilon BP gene is composed of six exons separated by five introns. The entire amino-terminal repetitive sequence is contained in exon III, and the carboxyl-terminal domain is encoded by the three succeeding exons (IV, V, VI). The latter three exons correspond well in size and share sequence homology with three exons coding for 14-kDa S-type lectins. The sequence in exon I offers an explanation for the generation of two mRNAs differing only in their 5' untranslated sequences, previously reported in Mac-2 cDNA clones. Using cDNA synthesis and PCR amplification, we determined that two alternative splice sites are used in many different types of cells. This alternative splicing results in different 5' untranslated regions of the murine epsilon BP mRNA.     

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.     

Cloning, mapping and genomic organization of a fish C-type lectin gene from homozygous clones of rainbow trout (Oncorhynchus mykiss)

Utilizing a splenic cDNA library and rapid amplification of cDNA 5' ends (5'-RACE), a C-type lectin gene was cloned from a homozygous cloned rainbow trout. The 1176 bp cDNA contains a 714 bp open reading frame from which a 238-amino-acid (aa) (27 kDa) protein was deduced. It was confirmed that this protein belongs to the C-type animal lectins, and is a type II membrane receptor. The predicted protein from this sequence contains a 48 aa cytoplasmic domain, a 20 aa transmembrane domain (TM), a 46 aa stalk region and a 124 aa carbohydrate-recognition domain (CRD). The stalk region contains a leucine-zipper, and an N-glycosylation site was also found in the CRD. Sequence alignment and phylogenetic analysis of the CRD indicate that the protein has similarity with human dendritic cell immunoreceptor (DCIR), gp120 binding C-type lectin (gp120BCL) and mammalian hepatic lectins. The N-terminus (aa 4-183) has similarity with NKG2, a group of C-type lectin receptors important in human natural killer cell function. The genomic DNA (gDNA) containing this gene was amplified and sequenced. The 4569 bp gDNA contains five exons and four introns. The first three exons encode the cytoplasmic domain, the TM and stalk region, respectively. Unlike the other type II C-type lectin receptors in which the CRD was encoded by three exons, the CRD of this lectin was encoded by two exons. A transposon Tc1-like fragment was found in intron III. Intron IV is composed of a simple repeat. Tissue-specific expression of the gene was studied by RT-PCR, and it was mainly expressed in spleen and peripheral blood leukocyte (PBL). Using AluI to digest the fragment containing exon I, intron I and exon II, an RFLP was produced between the sequences of this gene in two cloned fish, OSU 142 and Arlee (AR). Seventy-one doubled haploids (DH) of OSU X AR were screened, and the gene was mapped to linkage group XIV on the published map (Young et al., Genetics 148 (1998) 839).     

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.     

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.     

Characterization of a major histocompatibility class II A gene (Clha-DAA) with an embedded microsatellite marker in Atlantic herring (Clupea harengus L.)

An Atlantic herring major histocompatibility class II A ( Clha-DAA ) cDNA sequence has been characterized and was shown to encode a leader peptide, alpha-1 domain, alpha-2 domain, connecting peptide, transmembrane and cytoplasmic region. The Clha-DAA protein sequence has all the characteristics of a teleost class II A protein with conserved cysteines in both the alpha-1 and the alpha-2 domains and two potential N-linked glycosylation sites. Exon 2 sequences encoding the polymorphic alpha-1 domain from different individuals were analysed and revealed the presence of at least two loci. The Clha-DAA gene consists of four exons and three short introns. Four unique intron 3 sequences from multiple individuals were obtained and were shown to contain a (TG)_n microsatellite sequence. Primers were optimized such that only a single microsatellite locus designated Clha-DAA-INTR3 was amplified. Four herring populations from the North Sea and the Baltic Sea were genotyped for Clha-DAA-INTR3 . In total, 16 Clha-DAA-INTR3 alleles were detected; the distribution of the alleles showed no deviation from Hardy–Weinberg expectation. Levels of genetic differentiation among samples were of similar magnitude as have been reported earlier for neutral microsatellite loci between northern North Sea and Baltic Sea herring populations.     

Nucleotide sequence of a chicken delta-crystallin gene.

We have determined the complete nucleotide sequence of one of the two non-allelic delta-crystallin genes in the chicken, arbitrarily designated delta-gene 1, using a genomic clone (lambda g delta 106) containing the entire gene sequence. By comparison of the genomic sequence and the delta-crystallin cDNA sequence previously determined, we have identified exon sequences in the genomic sequence. Thus, the presence of 17 exons and 16 introns in the gene has been clarified. The delta-crystallin polypeptide deduced from the exon sequences consists of 465 amino acids which is larger, by 19 amino acid residues, than the polypeptide deduced from the cDNA sequence previously reported. Re-examination of the cDNA sequence using the same cDNA clone previously used shows that the present exon sequences are correct and the molecular weight of the deduced delta-crystallin polypeptide is 50,615 daltons instead of the previously reported value of 48,447 daltons. In addition, some structural features of the delta-crystallin gene including putative expression signals are discussed.     

Molecular characterization of the equine ATP2A2 gene

The mammalian ATP2A2 gene encodes a P-type cation pump located in the sarcoplasmic or endoplasmic reticula of muscle cells. We isolated one bacterial artificial chromosome (BAC) clone containing the equine ATP2A2 gene and determined the complete coding sequence of this gene. Cloning and characterization of the equine ATP2A2 gene revealed that the equine ATP2A2 gene consists of 20 exons. In total, 32 horses out of 16 breeds were analyzed for single nucleotide polymorphisms (SNPs). A mutation scan for SNPs included ten exons and their flanking introns. We detected in total 17 SNPs, 14 of which were located in introns, one in exon 9 and two in exon 20. In this report we provide the genomic organization and the equine ATP2A2 coding sequence and an association analysis for chronic pastern dermatitis using a sample of South German draft horses.     

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.     

The human XPG gene: gene architecture, alternative splicing and single nucleotide polymorphisms

Defects in the XPG DNA repair endonuclease gene can result in the cancer-prone disorders xeroderma pigmentosum (XP) or the XP-Cockayne syndrome complex. While the XPG cDNA sequence was known, determination of the genomic sequence was required to understand its different functions. In cells from normal donors, we found that the genomic sequence of the human XPG gene spans 30 kb, contains 15 exons that range from 61 to 1074 bp and 14 introns that range from 250 to 5763 bp. Analysis of the splice donor and acceptor sites using an information theory-based approach revealed three splice sites with low information content, which are components of the minor (U12) spliceosome. We identified six alternatively spliced XPG mRNA isoforms in cells from normal donors and from XPG patients: partial deletion of exon 8, partial retention of intron 8, two with alternative exons (in introns 1 and 6) and two that retained complete introns (introns 3 and 9). The amount of alternatively spliced XPG mRNA isoforms varied in different tissues. Most alternative splice donor and acceptor sites had a relatively high information content, but one has the U12 spliceosome sequence. A single nucleotide polymorphism has allele frequencies of 0.74 for 3507G and 0.26 for 3507C in 91 donors. The human XPG gene contains multiple splice sites with low information content in association with multiple alternatively spliced isoforms of XPG mRNA.