Structure and genomic organization of the mouse dihydrofolate reductase gene

The genomic organization of the mouse dihydrofolate reductase gene has been determined by hybridization of specific cDNA sequences to restriction endonuclease-generated fragments of DNA from methotrexate-resistant S-180 cells. The dihydrofolate reductase gene contains a minimum of five intervening sequences (one in the 5′ untranslated region and four in the protein-coding region) and spans a minimum of 42 kilobase pairs on the genome. Genomic sequences at the junction of the intervening sequence and mRNA-coding sequence and at the polyadenylation site have been determined. A similar organization is found in independently isolated methotrexate-resistant cell lines, in the parental sensitive cell line and in several inbred mouse strains, indicating that this organization represents that of the natural gene.     

Structural organization of the mouse lactoferrin gene.

The complete structure of the mouse lactoferrin gene is presented. Mouse lactoferrin (mLF) is encoded by a single copy gene of approximately 30 kilobases (kb) in size. The gene is organized into 17 exons separated by 16 introns. The exons range in size from 48 base pairs (bp) to 190 bp whereas the introns range from 0.2 kb to 4.3 kb. Structural analysis of the mouse lactoferrin gene reveals that this gene shares a similar intron-exon distribution pattern with both human transferrin and chicken ovotransferrin.     

Structure and organization of the murine band 3 gene

The Band 3 protein mediates the reversible exchange of chloride and bicarbonate anions across the plasma membrane of erythrocytes, and probably, certain epithelial cells. It also serves to anchor the spectrin cytoskeleton to the plasma membrane via its association with ankyrin. We have isolated and largely sequenced the 17-kilobase murine Band 3 gene. We show that this gene is present in a single copy in the mouse genome and have identified and mapped the 19 intervening sequences. The locations of the intron/exon junctions in the Band 3 mRNA correlate with predicted structural features of the erythrocyte Band 3 protein structure and membrane topology. One of the introns within this gene contains a single copy of a murine Alu-type high dispersed sequence, in addition to several unusual tandemly repeated sequences.     

Genomic organization of the mouse OSF-1 gene.

The mouse OSF-1 protein (also known as pleiotrophin, HB-GAM, HBGF-8, or HBNF) gene was isolated from a mouse genomic library and sequenced. OSF-1 is a 15-kD secreted protein specifically expressed in bone and brain, and is believed to play a role in brain development and osteogenesis. The mouse OSF-1 gene consists of at least 5 exons and 4 introns and spans > 32 kb. Computer analysis of approximately 4 kb of 5'-flanking sequence of the OSF-1 gene revealed two candidate promoter regions. One candidate promoter contains a thyroid hormone/retinoic acid-responsive element and the other contains two glucocorticoid-responsive elements. DNA sequence analysis of novel OSF-1 cDNA clones indicates that two promoters can be utilized in MC3T3-E1 osteoblastic cells. The overall organization of the mouse OSF-1 gene is similar and the locations of the three exon-intron junctions within the coding region are identical to the mouse gene encoding the differentiation-related factor midkine (MK). Based on this similarity and on the high degree of nucleotide sequence homology (approximately 55%) of mouse OSF-1 and mouse MK, we conclude that OSF-1 and MK are generated from a common ancestral gene and are members of a family of structurally and probably functionally related proteins.     

Different structural organization of the encephalopsin gene in man and mouse

Encephalopsin, also called Panopsin, is a recently discovered extraretinal photoreceptor, which may play a role in non-visual photic processes such as the entrainment of circadian rhythm or the regulation of pineal melatonin production. Based on RT-PCR data and comparative genomic sequence analysis, we show that the human OPN3 gene consists of six exons and expresses various splice variants, while the murine homologue contains four exons and produces just one splice form. Furthermore, the human OPN3 gene overlaps with the neighboring KMO gene on a genomic as well as on an RNA level, whereas the corresponding genes in mouse lie close together but do not overlap. This finding is of particular interest, since differences in gene organization between man and mouse, that have been reported so far, occur within gene clusters, i.e. the number of genes within a certain cluster may differ between man and mouse. OPN3 provides an exception to this rule, since it is positionally uncoupled from other genes of the opsin family.     

Genomic organization and chromosomal localization of the mouse IKBKAP gene.

The autosomal recessive disorder familial dysautonomia (FD) has recently been demonstrated to be caused by mutations in the IKBKAP gene, so named because an initial report suggested that it encoded an IkappaB kinase complex associated protein (IKAP). Two mutations in IKBKAP have been reported to cause FD. The major mutation is a T-->C transition in the donor splice site of intron 20 and the minor mutation is a missense mutation in exon 19 that disrupts a consensus serine/threonine kinase phosphorylation site. We have characterized the cDNA sequences of the mouse, rat and rabbit IKBKAP-encoded mRNAs and determined the genomic organization and chromosomal location of mouse IKBKAP. There is significant homology in the amino acid sequence of IKAP across species and the serine/threonine kinase phosphorylation site altered in the minor FD mutation of IKAP is conserved. The mouse and human IKBKAP genes exhibit significant conservation of their genomic organization and the intron 20 donor splice site sequence, altered in the major FD mutation, is conserved in the human and mouse genes. Mouse IKBKAP is located on the central portion of chromosome 4 and maps to a region in which there is conserved linkage homology between the human and mouse genomes. The homologies observed in the human and mouse sequences should allow, through the process of homologous recombination, for the generation of mice that bear the IKBKAP mutations present in individuals with FD. The characterization of such mice should provide significant information regarding the pathophysiology of FD.     

Exon-intron organization and chromosomal localization of the mouse monoglyceride lipase gene

Monoglyceride lipase (MGL) functions together with hormone-sensitive lipase to hydrolyze intracellular triglyceride stores of adipocytes and other cells to fatty acids and glycerol. In addition, MGL presumably complements lipoprotein lipase in completing the hydrolysis of monoglycerides resulting from degradation of lipoprotein triglycerides. Cosmid clones containing the mouse MGL gene were isolated from a genomic library using the coding region of the mouse MGL cDNA as probe. Characterization of the clones obtained revealed that the mouse gene contains the coding sequence for MGL on seven exons, including a large terminal exon of approximately 2.6 kb containing the stop codon and the complete 3' untranslated region. Two different 5' leader sequences, diverging 21 bp upstream of the predicted translation initiation codon, were isolated from a mouse adipocyte cDNA library. Western blot analysis of different mouse tissues revealed protein size heterogeneities. The amino acid sequence derived from human MGL cDNA clones showed 84% identity with mouse MGL. The mouse MGL gene was mapped to chromosome 6 in a region with known homology to human chromosome 3q21.     

Structure and organization of the human transglutaminase 1 gene.

Membrane-associated transglutaminases (TGase1) have recently been found to be common in mammalian cells, but it is not clear whether these derive from the same or different genes. In order to determine the complexity of this system, we have isolated and characterized the human gene (TGM1). The gene of 14,133 base pairs was found to contain 15 exons spliced by 14 introns. Interestingly, the positions of these introns have been conserved in comparison with the genes of two other transglutaminase-like activities described in the literature, but the TGM1 gene is by far the smallest characterized to date because its introns are relatively smaller. On the other hand, the TGase1 enzyme is the largest known transglutaminase (about 90 kDa), apparently because its gene acquired tracts that encode additional sequences on its amino and carboxyl termini that confer its unique properties. Southern blot analyses of total human genomic DNA cut with several restriction enzymes reveal only one band. Use of human-rodent cell hybrid panels and chromosomal in situ hybridization with biotin-labeled probes revealed that the human TGM1 gene maps to chromosome position 14q11.2-13. Such data suggest there is a single gene copy per haploid human genome. Comparisons of sequence identities and homologies indicate that the transglutaminase family of genes arose by duplications and subsequent divergent evolution from a common ancestor but later became scattered in the human genome. Although our present Southern blot and chromosomal localization studies revealed no restriction fragment length polymorphisms, comparisons of published sequences and our genomic clone indicate there are two sequence variants for TGase1 within the human population. The rare smaller variant contains a two-nucleotide deletion near the 5'-end, uses an alternate initiation codon, and differs from the common larger variant only in the first 15 amino acids. Furthermore, the DNA sequences of intron 14 possess several tracts of dinucleotide repeats that by polymerase chain reaction analysis show wide size polymorphism within the human population. Accordingly, this gene system constitutes a useful polymorphic marker for genetic linkage analyses.     

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.