The cDNA structure, expression, and chromosomal assignment of the mouse Fas antigen.

The cell surface Fas antigen is a membrane-associated polypeptide which can mediate apoptosis. cDNA clones encoding the Fas antigen were isolated from a cDNA library constructed with mRNA from the mouse macrophage cell line BAM3. The nucleotide sequence and the deduced amino acid sequence of the mouse Fas antigen were 58.5 and 49.3% identical, respectively, to the corresponding sequences of human Fas antigen cDNA. The mouse Fas antigen consists of 306 amino acids with a calculated Mr of 34,971 and contains a single transmembrane domain which divides the molecule into extracellular and cytoplasmic domains. A 2.1-kb mRNA coding for the Fas antigen was detected in the mouse thymus, heart, liver, and ovary but not in brain and spleen. The expression of the Fas antigen gene in mouse fibroblast L929 and macrophage BAM3 cell lines was significantly induced by treatment with IFN-gamma but not by IFN-alpha/beta. Interspecific backcross analysis indicated that the gene coding for the Fas antigen is in the distal region of mouse chromosome 19.     

Molecular isolation and sequence determination of the cDNA for the mouse sperm-specific lactate dehydrogenase-X gene

Several cDNA clones for the mouse lactate dehydrogenase-X (LDH-X), a sperm-specific glycolytic enzyme, were isolated from mouse testicular cDNA libraries constructed in the bacteriophage vectors, lambda gt11 and gt10. The largest cDNA clone contains an insert of 1135 base pairs in length and an open reading frame that encodes a 332 amino acid polypeptide with a molecular weight of 35.89 kD. The deduced amino acid sequence of this protein is in close agreement with the published sequence of mouse LDH-X obtained by direct protein sequencing. Northern analysis of RNA isolated from different tissues detected a single size mRNA of 1.5 kilobases in mouse testis but not in brain or liver. The Ldh-x structural gene was estimated to be about 12 kb in size as demonstrated by Southern hybridization analysis of mouse genomic DNA using the full-length cDNA as a probe.     

Structure and tissue distribution of a trinucleotide-repeat-containing gene (cag-3) expressed specifically in the mouse brain

Using in silico approaches and RACE we cloned a full length trinucleotide (CAG) repeat-containing cDNA (cag-3). The cDNA is 2478 bp long and the deduced polypeptide consists of 140 amino acids of which 73 are glutamines. The genomic sequence spans approximately 79 kb on mouse chromosome 7 and the gene is composed of four exons. Standard and real-time PCR analyses of several mouse tissues showed that the gene is exclusively expressed in the brain and is not detected in embryonic stages. Within the brain, it is expressed throughout the forebrain region with predominant expression in the hypothalamus and olfactory bulb and very low levels in the mid- and hindbrain.     

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     

Profiles of PrKX expression in developmental mouse embryo and human tissues.

Protein kinase X (PrKX), karyotypically located on the human X chromosome, is a type I cAMP-dependent protein kinase. Although a specific role for PrKX has not yet been defined, PrKX gene expression in mouse and human tissues has been profiled only by in situ hybridization and Northern blot analyses and not by protein expression. To determine more precisely the PrKX protein levels, we developed specific anti-PrKX antibodies and examined gestationally staged mouse embryo sections by immunohistochemistry. These results showed that PrKX is ubiquitously distributed and highly expressed in murine central nervous system and heart tissues in early developmental stages and in most organs at later stages but was not detected in either connective tissues or bone. Using Western blots to detect PrKX, total protein extracts from eight different adult or fetal human tissues including brain, heart, kidney, liver, lung, pancreas, spleen, and thymus were analyzed. Although PrKX protein was present in each of the tissues tested, the protein levels varied depending on tissue type and developmental stage. Very low protein levels were found in heart tissues from a 5-month-old fetus and from an adult, whereas PrKX proteins were more abundant in fetal brain, kidney, and liver tissues compared with adult samples of the same tissue type.     

Glucose transporter expression in brain. cDNA sequence of mouse GLUT3, the brain facilitative glucose transporter isoform, and identification of sites of expression by in situ hybridization.

Complementary DNAs encoding the mouse GLUT3/brain facilitative glucose transporter have been isolated and sequenced. The predicted amino acid sequence indicates that mouse GLUT3 is composed of 493 amino acids and has 83 and 89% identity and similarity, respectively, to the sequence of human GLUT3. In contrast to human GLUT3 mRNA, which can be readily detected by RNA blotting in all human tissues that have been examined, mouse GLUT3 mRNA was only present at significant levels in brain. In situ hybridization showed differential expression of GLUT3 mRNA in several regions of adult mouse brain. Specific expression was observed in the hippocampus, with GLUT3 mRNA levels being higher in areas CA1 to CA3 than in the dentate gyrus. It was also detected in the Purkinje cell layer of the cerebellum and in the cerebral cortex, with higher expression in the piriform cortex than in other regions of the cortex. Antisera to mouse GLUT3 immunoblotted a series of proteins of 45-50 kDa in mouse brain plasma membranes. These results are consistent with GLUT3 being a neuronal glucose transporter.     

Regional assignment of ADA and ITPA to mouse chromosome 2 (C1----ter). A demonstration of the conserved linkage of enzyme and proto-oncogene loci

Similarity of G-band patterns between the long arm of Chinese hamster chromosome 6 and mouse chromosome 2, combined with the assignments of AK1, ADA, and ITPA to hamster chromosome 6 and AK1 to mouse chromosome 2, suggested mouse chromosome 2 also might contain ADA and ITPA. Here, concordant segregation analysis of enzyme loci and chromosomes in mouse spleen X CHO as well as mouse microcell X CHO somatic cell hybrids established the assignments of ADA and ITPA onto mouse chromosome 2 in the region between the first G-band and the terminus (C1----ter). This assignment presents a demonstration of the conservation and evolution of enzyme and proto-oncogene loci linkage since two cellular homologs of viral oncogenes--c-src and c-abl--also map to mouse chromosome 2. In humans c-src, ADA, and ITPA remain conserved on chromosome 20, whereas AK1 and c-abl are together on chromosome 9. These observations and concepts are discussed with respect to the role of proto-oncogenes in chromosomal evolution and suggest the long arm of chromosome 6 as a fruitful place to look for c-src and c-abl in the Chinese hamster.     

Characterization of the mouse hnRNP A2/B1/B0 gene and identification of processed pseudogenes

The mouse hnRNP A2/B1/B0 gene has been cloned using a PCR-based strategy and sequenced. Analysis of this sequence showed that the gene organization closely follows that of the human orthologue with 12 exons and 11 introns. The hnRNP A2/B1/B0 gene gives rise to four splice variants through alternative splicing of exons 2 and 9. RT-PCR assays indicated that all splice variants were expressed in mouse brain, skin, and stomach tissues of varying ages, although their ratios to one another varied with age and tissue type. We also identified a small subset of all polyadenylated splice variants that included intron 11, which shows 94% sequence identity between human and mouse. Several processed pseudogenes were identified in the mouse genome. A search of the mouse genome databases located five pseudogenes, four of which are presumed to be non-functional because of the presence of premature stop codons, large deletions or rearrangements within the coding region. The fifth, which possesses putative promoter elements and has a coding sequence identical to that of the hnRNP A2 mRNA variant, may be functional.     

Mouse and human GTPBP2, newly identified members of the GP-1 family of GTPase

We earlier identified the GTPBP1 gene which encodes a putative GTPase structurally related to peptidyl elongation factors. This finding was the result of a search for genes, the expression of which is induced by interferon-gamma in a macrophage cell line, THP-1. In the current study, we probed the expressed sequence tag database with the deduced amino acid sequence of GTPBP1 to search for partial cDNA clones homologous to GTPBP1. We used one of the partial cDNA clones to screen a mouse brain cDNA library and identified a novel gene, mouse GTPBP2, encoding a protein consisting of 582 amino acids and carrying GTP-binding motifs. The deduced amino acid sequence of mouse GTPBP2 revealed 44.2% similarity to mouse GTPBP1. We also cloned a human homologue of this gene from a cDNA library of the human T cell line, Jurkat. GTPBP2 protein was found highly conserved between human and mouse (over 99% identical), thereby suggesting a fundamental role of this molecule across species. On Northern blot analysis of various mouse tissues, GTPBP2 mRNA was detected in brain, thymus, kidney and skeletal muscle, but was scarce in liver. Level of expression of GTPBP2 mRNA was enhanced by interferon-gamma in THP-1 cells, HeLa cells, and thioglycollate-elicited mouse peritoneal macrophages. In addition, we determined the chromosomal localization of GTPBP1 and GTPBP2 genes in human and mouse. The GTPBP1 gene was mapped to mouse chromosome 15, region E3, and human chromosome 22q12-13.1, while the GTPBP2 gene is located in mouse chromosome 17, region C-D, and human chromosome 6p21-12.     

cDNA cloning, characterization and chromosome mapping of Crtap encoding the mouse cartilage associated protein.

Recently we have isolated and characterized a cDNA coding for a novel developmentally regulated chick embryo protein, cartilage associated protein (CASP). Here we describe the isolation and characterization of the cDNAs coding for the mouse CASP. Comparison of the mammalian putative protein sequence with the chick sequence shows a very high identity overall (51%); in particular the chick protein is homologous to the half amino terminus of the mouse protein. Furthermore, the comparison of the CASP cDNA sequence with sequences of the genebank database confirms our hypothesis that the CASP genes belong to a novel family that also includes genes encoding for some nuclear antigens. In all mouse tissues examined three CASP mRNAs species are detected, whereas in chick tissues a single mRNA is present. Immunohistochemistry studies show that the protein is expressed in all mouse embryonic cartilages. The mouse cartilage associated protein gene (Crtap) was assigned to chromosome 9F3-F4 by fluorescence in situ hybridization.