Sequence of the gene for the constant region of the mu chain of Balb/c mouse immunoglobulin

We present the complete sequence of Cμ immunoglobulin constant region gene of mouse with 5′ flanking and 3′ untranslated regions. The gene consists of four exons coding for protein domains which are separated by three introns. Intensive study of the homology relationship of DNA sequences within the Cμ gene and Cγ2b gene leads us to believe that the shifting of existing splice sites along with creation of new ones plays a significant role in evolution, driving the reversible reaction exon ? intron.     

Isolation of the osteonectin gene: evidence that a variable region of the osteonectin molecule is encoded within one exon

A complementary DNA clone for bovine osteonectin was used to isolate the osteonectin gene from two libraries of bovine genomic DNA fragments. Two overlapping clones were obtained whose relationship was determined by restriction mapping and sequence analysis. The two clones contain the entire osteonectin coding region spanning approximately 11 kilobases of genomic DNA. The coding region of the gene was determined, by electron microscopy and DNA sequencing, to reside in nine exons. In addition, there is at least one 5' exon interrupted by an intron in the 5'-nontranslated sequence of the gene. Excluding this 5' exon and the 3'-terminal exon, the exons are small and approximately uniform in size, averaging 130 +/- 17 base pairs. Three of the exons at the 5' end of the gene were sequenced and appear to encode discrete protein domains. For example, the putative exon 2 contains the coding region for the leader peptide of the molecule. The amino-terminal protein sequence was determined for osteonectin extracted from human, rabbit, and chicken bone and compared with those for bovine, mouse, and pig osteonectin. These data suggest that osteonectin is highly conserved between species, interspecies changes being seen primarily at the amino terminus of the protein and specifically in the region encoded by putative exon 3 in the bovine gene.     

Structure of the rat alpha 2-macroglobulin-coding gene

Rat alpha 2-macroglobulin (alpha 2M) is an acute-phase protein, i.e., produced upon tissue inflammation. Genomic DNA clones covering the entire sequence of the alpha 2M gene were isolated and characterized by restriction mapping. Southern blotting and (partial) DNA sequencing. The rat alpha 2M gene is approx. 50 kb in length and consists of 36 exons ranging in size from 21 to 229 bp. Two functional domains, a bait region and a thiol ester site, are encoded by the exon 18 and 24, respectively. Several possible regulatory signals such as a TPA-inducible enhancer core, an identifier sequence, purine-pyrimidine alternative stretches and viral enhancer core sequences were identified. Several genomic DNA clones which cross-hybridized with the alpha 2M cDNA probe were also identified. Sequence analysis showed that they possessed sequences identical to a part of the rat alpha 1-inhibitor III cDNA and that they had a strikingly similar exon organization to the alpha 2M gene.     

The Structure and Organization of the Human Erythroid Anion Exchanger (AE1) Gene

The AE1 (anion exchanger, band 3) protein is expressed in erythrocytes and in the A-type intercalated cells of the kidney distal collecting tubule. In both cell types it mediates the electroneutral transport of chloride and bicarbonate ions across the lipid bilayer, and, in erythrocytes, it also serves as the critical attachment site of the peripheral membrane skeleton. We have characterized the human AE1 gene using overlapping clones isolated from a phage library of human genomic DNA. The gene spans 20 kb and consists of 20 exons separated by 19 introns. The structure of the human AE1 gene corresponds closely with that of the previously characterized mouse AE1 gene, with a high degree of conservation of exon/intron junctions, as well as exon and intron nucleotide sequences. The putative upstream and internal promoter sequences of the human AE1 gene used in erythroid and kidney cells, respectively, are described. We also report the nucleotide sequence of the entire 3′ noncoding region of exon 20, which was lacking in the published cDNA sequences. In addition, we have characterized 9 Alu repeat elements found within the body of the human AE1 gene that are members of 4 related subfamilies that appear to have entered the genome at different times during primate evolution.     

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.     

A genetic polymorphism in a functional domain of human pregnancy zone protein: the bait region. Genomic structure of the bait domains of human pregnancy zone protein and alpha 2 macroglobulin.

Genomic clones containing the exons coding for the bait domain of human pregnancy zone protein and alpha 2 macroglobulin were isolated and fragments containing the bait exons were sequenced. It is shown that the bait domains of both alpha 2 macroglobulin and pregnancy zone protein are encoded by two exons, with conserved exon/intron boundaries. A genetic polymorphism showing either a Met or a Val residue as the sixth amino acid of the pregnancy zone protein bait domain was detected with the rare Met allele showing a gene frequency of 0.065.     

Genomic structure and chromosomal mapping of the murine CD40 gene.

The B cell-associated surface molecule, CD40, is likely to play a central role in the expansion of Ag-stimulated B cells, and their interaction with activated Th cells. In our study we have isolated genomic clones of murine CD40 from a mouse liver genomic DNA library. Comparison with the murine CD40 cDNA sequence revealed the presence of nine exons that together contain the entire murine CD40 coding region, and span approximately 16.3 kb of genomic DNA. The intron/exon structure of the CD40 gene resembles that of the low affinity nerve growth factor receptor gene, a close homolog of both human and murine CD40. In both cases the functional domains of the receptor molecules are separated onto different exons throughout the genes. Southern blot analysis demonstrated that murine CD40 is a single copy gene that maps in the distal region of mouse chromosome 2.     

Structure and expression of the mouse prealbumin gene

We cloned a genomic DNA fragment which covers the entire sequence of the mouse prealbumin gene and then studied the structure. The coding regions are separated into four exons by three introns, and these numbers, the sizes of the exons and the relative sites of the exon-intron junctions are all in complete agreement with those determined for the human gene. The sequences of four exons can be aligned perfectly with that of the previously determined mouse prealbumin cDNA. In addition to the exon regions, we found two highly conserved DNA regions between the mouse and human prealbumin genes, one in the 5'-flanking region of the gene and the other in the 3' end region of the first intron. These DNA regions contain several consensus glucocorticoid receptor-binding site sequences, and the latter also contains an enhancer sequence present in the immunoglobulin kappa-chain joining-constant kappa intron. RNA hybridizing to the mouse prealbumin cDNA was detected in the extracts from liver, brain, and kidney, but was not detected in testes, spleen, or heart. Little change was caused in the level of prealbumin mRNA in the liver by administration of dexamethasone to mice.     

Genomic Structure and Chromosomal Localization of Mouse Cyclin G1 Gene

Mouse genomic DNA harboring the full coding sequence of cyclin G1 was cloned and analyzed. The locations of five coding exons and the intron–exon boundary sequences were found to be conserved between the mouse and the human genes. Two putative binding sites for thep53tumor suppressor gene product were found around the first exon: one was located in the 5′ regulatory region, and the other was in the first intron. The mouse cyclin G1 gene was mapped to bands A5 to B1 of chromosomes 11 (11A5–B1) by FISH using genomic DNA clone as a biotinylated probe. The location of mouse cyclin G1 is syntenic to that of its human homologue, which we previously mapped to 5q32–q34 of chromosome 5. An additional faint signal was detected on chromosome 4 (4B1–C2), probably indicating the presence of a cyclin G1-related gene or pseudogene in the mouse genome.     

The human neutrophil elastase gene. Analysis of the nucleotide sequence reveals three distinct classes of repetitive DNA

DNA sequence analysis reveals the gene encoding human neutrophil elastase to be contained on a 6-kb EcoRI fragment. The gene contains five exons and closely resembles rat mast cell proteinase II and mouse adipsin in its exon structure and intron splice phase. Non-coding regions are very rich in repetitive DNA, containing seven Alu-like segments, three distinct clustered direct repeats with monomer lengths of 53 (six repeats), 23 (three repeats) and 41 (ten repeats) nucleotides, and a 200-nucleotide AT-rich region. Protein sequence analysis, inferred from the coding regions of the gene, indicates that neutrophil elastase may contain an unusual activation peptide similar to that found in the other major neutrophil serine proteinase, cathepsin G.     

Mouse connexin 45: genomic cloning and exon usage

Connexin 45 is a gap junction protein that is prominent in early embryos and is widely expressed in many mature cell types. To elucidate its gene structure, expression, and regulation, we isolated mouse Cx45 genomic clones. Alignment of the genomic DNA and cDNA sequences revealed the presence of three exons and two introns. The first two exons contained only 5' untranslated sequences, while exon 3 contained the remaining 5' UTR, the entire coding region, and the 3' UTR. An RT-PCR with exon-specific primers was utilized to examine exon usage in F9 mouse embryonal carcinoma cells and adult mouse tissues. In all samples, PCR products amplified using exon 2/exon 3 or exon 3/exon 3 primer pairs were much more abundant than products produced using exon 1/exon 2 or exon 1/exon 3 primer pairs, suggesting that Cx45 mRNAs containing exon 1 were relatively rare compared with mRNAs containing the other exons. Rapid amplification of cDNA ends (5'-RACE) was performed using antisense primers from within exon 3 and template RNA prepared from F9 cells or from adult mouse kidney. We obtained multiple RACE products from both templates, including products that contained all three exons and were spliced identically to the cDNA. However, clones were also isolated (from kidney) that began within the region previously identified as intron 1 and continued upstream with a sequence identical to the cDNA, including splicing to exon 3. These results show that mouse Cx45 has a gene structure that differs from that of previously studied connexins and allows the production of heterogeneous Cx45 mRNAs with differing 5' UTRs. These differences might contribute to regulation of Cx45 protein levels by modulating mRNA stability or translational efficiency.     

Ovoinhibitor introns specify functional domains as in the related and linked ovomucoid gene

We have isolated cDNA clones and determined the gene structure of chicken ovoinhibitor, a seven domain Kazal serine proteinase inhibitor. Using RNA blot hybridization analysis, the gene was identified initially as a region 9-23 kilobases upstream of the gene for the related inhibitor ovomucoid. Ovoinhibitor RNA appears in oviduct and liver. cDNA clones were identified by screening an oviduct cDNA library with a nick-translated DNA restriction fragment which contained an exon of the gene. The mature protein sequence derived from a cDNa clone is in excellent agreement with that which we obtained from direct sequencing of purified ovoinhibitor. The protein-sequencing strategy is reported. The P1 amino acids of the Kazal domains are consistent with the known broad inhibitory specificity of ovoinhibitor. The gene is about 10.3 kilobases in length and consists of 16 exons. Each Kazal domain is encoded by two exons. Like ovomucoid, introns fall between the coding sequences of the ovoinhibitor domains, an arrangement which may have facilitated domain duplication. The intradomain intron occurs in an identical position in all of the ovoinhibitor and ovomucoid Kazal domains, suggesting that this intron was present in the primordial inhibitor gene. We discuss the location of the intradomain intron in relation to the known structure of four Kazal inhibitors and suggest a scheme for the evolution of the ovoinhibitor gene.