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.     

Complete primary structure and genomic organization of the mouse Col14a1 gene.

The entire mouse cDNA sequence for type XIV collagen was determined using overlapping PCR products. The 6456 nucleotide (nt) cDNA sequence contains a 5391-nt open reading frame encoding 1797 amino acid residues. The amino terminus has a 28-residue signal peptide that is followed by the mature polypeptide of 1769 amino acid residues with a calculated molecular mass of 193.2 kDa. The mouse alpha1(XIV) collagen chain is predicted to contain all the structural domains described for the polypeptide in chicken and human. These include fibronectin type III repeats, von Willebrand factor A domains, thrombospondin-N-terminal-like domains and two triple-helical domains similar to those of other collagen family members. The amino acid residue sequence of human alpha1(XIV) collagen showed an overall identity of 74% to the chicken sequence and 88% to the human sequence. The entire mouse genomic structure has been determined and is made up of 48 exons. Alternatively spliced forms of mouse type XIV, collagen were not identified corresponding to the findings for the human form.     

Complete primary structure and genomic organization of the mouse Col14a1 gene.

The entire mouse cDNA sequence for type XIV collagen was determined using overlapping PCR products. The 6456 nucleotide (nt) cDNA sequence contains a 5391-nt open reading frame encoding 1797 amino acid residues. The amino terminus has a 28-residue signal peptide that is followed by the mature polypeptide of 1769 amino acid residues with a calculated molecular mass of 193.2 kDa. The mouse alpha1(XIV) collagen chain is predicted to contain all the structural domains described for the polypeptide in chicken and human. These include fibronectin type III repeats, von Willebrand factor A domains, thrombospondin-N-terminal-like domains and two triple-helical domains similar to those of other collagen family members. The amino acid residue sequence of human alpha1(XIV) collagen showed an overall identity of 74% to the chicken sequence and 88% to the human sequence. The entire mouse genomic structure has been determined and is made up of 48 exons. Alternatively spliced forms of mouse type XIV, collagen were not identified corresponding to the findings for the human form.     

Structure and genomic organization of porcine RACK1 gene

The cDNA encoding porcine RACK1 protein was isolated from porcine spleen cDNA library. The deduced protein sequence of porcine RACK1 cDNA shows that it contains 317 amino acid residues, and shares nearly 100% identity with its vertebrate counterparts. Noticeably, the RACK1 protein was differentially expressed in various porcine tissues. High expression of RACK1 protein was observed in the tissues including thymus, pituitary, spleen and liver, whereas there was no detectable expression in muscle. The genomic DNA of porcine RACK1 with approximate 7.5 kb was constructed by both polymerase chain reaction amplification and genomic library screening. It consists of eight exons intervened by seven introns, and most of the intron/exon splice sites conform to the GT/AG rule. The promoter region contains functional serum response element, YY1-like binding site and AP1 site, which is supported by the finding that the expression of RACK1 gene in cultured porcine ST cells has a serum response as well as a TPA response.     

Cloning,expression, and genomic organization of mouse mp29 gene

The convulsions of approximately 25% of epileptics are inadequately controlled by currently available medication; therefore the preparation of new antiepileptic drugs is of great interest. Aryl semicarbazones can be considered a new class of compounds presenting anticonvulsant activity. In addition, they can be orally administered and are more active as anticonvulsants than mephenytoin or phenobarbital. However, one disadvantage of these compounds is their low water solubility. As a strategy to circumvent this problem, a 1:1 inclusion compound of benzaldehyde semicarbazone (BS) and hydroxypropyl-beta-cyclodextrin (HP-beta-CD) was prepared and characterized. The anticonvulsant activities of the free semicarbazone and of the inclusion compound were evaluated in rats using the maximum electroshock and audiogenic seizures screenings. In both tests the minimum dose of compound necessary to produce activity decreases from 100mg/kg for the free semicarbazone to 35 mg/kg for the inclusion compound, indicating a significant increase in the bio-availability of the drug.     

Cloning and genomic organization of the mouse gene slc23a1 encoding a vitamin C transporter.

Vitamin C is known to exist in particularly high concentrations in brain tissue, and its free radical scavenging function is thought to represent a major antioxidative defense system. We have cloned, sequenced and analyzed the genomic structure of a mouse sodium-dependent vitamin C transporter gene, Slc23a1 (also known as Svct2). The mouse Slc23a1 cDNA is 6.4 kb long and was cloned directly from a mouse brain RNA preparation. Hybridization screening of a mouse genomic BAC library identified BAC 53L21 which contains at least the entire coding sequence of the mouse Slc23a1 gene. Determination of the exon-intron structure of the gene revealed 17 exons ranging from 58 bp to 4407 bp extending over 50 kb of the mouse genome, with the translation start codon located in exon 3. Its 1944 nucleotide open reading frame encodes a polypeptide of 647 aa, which is highly similar to rat and human orthologs. The mouse gene was assigned to chromosome 2qG2 by fluorescence in situ hybridization analysis. Expression of this gene was demonstrated in a wide range of tissues, with especially high levels in brain. Neurodegenerative diseases with an established role for oxidative stress in the cytoplasm may therefore be conditions of SLC23A1 dysfunction. Key words: gene structure; Vitamin C; transporter; oxidative stress     

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.     

Sequence, genomic organization and functional expression of the murine tRNA-specific adenosine deaminase ADAT1

We have recently identified the first mammalian tRNA-specific adenosine deaminase human ADAT1, a member of the ADAR family of RNA editing enzymes. This protein is responsible for the first step of the unique A(37) to m(1)I(37) modification in eukaryotic tRNA(Ala). Here, we present the genomic structure of murine ADAT1 and the functional expression of mADAT1 cDNA. In mouse, as well as in human, ADAT1 is expressed from a single copy gene. The coding region of the mADAT1 gene is spread over nine exons, covering approximately 30kb of genomic DNA and encodes a protein of 499 amino acids. Overall, mADAT1 shares 81% nucleotide homology and 87.5% protein homology with the human ortholog. The recombinant mouse protein is active specifically and with a high efficiency on human tRNA(Ala) in vitro. Its genomic organization is compared to the structures of the sequence-related, pre-mRNA specific adenosine deaminases ADAR1 and ADAR2.     

The genomic organization,promoter position and expression profile of the mouse MRG15 gene

MORF4 (mortality factor on chromosome 4) and the novel related MRG (MORF4-related gene) gene family were identified when MORF4 was shown to induce senescence in a subset of tumor cell lines. The gene on chromosome 15 (MRG15) has high similarity to Drosophila MSL3, which is a component of the dosage compensation complex. MRG15 also has a chromodomain and may therefore function as a chromatin remodeling factor in a complex(es) involving a histone acetyltransferase, similar to MSL3. To complement our studies on human MRG15, we cloned and characterized the mouse MRG15 gene. Mouse MRG15 is expressed ubiquitously in adult tissues and at various embryonic stages, and expression in adult testis is higher than in other tissues. MRG15-b, which is an alternatively spliced form of MRG15-a and has a 39-amino-acid insertion in the chromodomain, is also expressed in all mouse tissues examined and localizes to the nucleus of cells. It is possible that MRG15-b may lack the function of the chromodomain because of the additional amino acids and could potentially be the equivalent of the human MORF4 in the mouse. The mouse MRG15 gene is composed of twelve exons and spans over 24 kb DNA. Using luciferase constructs we have determined that there is a functional promoter sequence 1.8 kb upstream of the ATG start codon. This region contains no TATA box but has GC-rich regions, consistent with the ubiquitous expression we have observed.     

In vivo expression and genomic organization of the mouse cyclin I gene (Ccni)

Cyclins control cell-cycle progression by regulating the activity of cyclin-dependent kinases. Cyclin I was recently added to the cyclin family of proteins because of the presence of a cyclin box motif in the deduced amino-acid sequence. Cyclin I may share functional roles with cyclin G1 and G2 because of the high structural similarity between their deduced amino-acid sequences. However, the biological and functional roles of this subclass of cyclins remain obscure. The mouse cyclin G1 and G2 genes have previously been cloned and characterized. In this report, we describe the cloning of the mouse homolog of cyclin I. The cyclin I cDNA sequence was used to determine the genomic organization of the mouse cyclin I gene which co-localizes with cyclin G2 to chromosome 5E3.3-F1.3. Cyclin I was transcribed from seven exons distributed over more than 19kb of genomic sequence. The expression of cyclin I was determined in various tissues, but no clear correlation with the proliferative state was found. Furthermore, in contrast to cyclin G1, cyclin I expression was stable during cell-cycle progression after partial hepatectomy in both the absence and presence of DNA damage. Transient expression of cyclin I-green fluorescent protein (GFP) fusion proteins in cell lines showed that cyclin I was distributed throughout the cell in contrast with the mainly cytoplasmic localization of cyclin G2 and nuclear localization of cyclin G1. Our results indicate that despite the close structural similarity between cyclin G1, G2 and I, these three proteins are likely to have distinct biological roles.