A highly conserved major histocompatibility complex class I-related gene in mammals

We report here a cDNA sequence of a murine homolog of the human major histocompatibility complex (MHC) class I-related gene, MR1. The analyses revealed unprecedentedly high conservation of MR1 in the alpha1 and alpha2 domains (corresponding to the peptide-binding domains in the classical MHC class I molecules) between human and mouse (predicted amino acid identity: 90 and 89% for the alpha1 and alpha2 domain, respectively), compared to MHC class I and other class I molecules. On the other hand, conservation in the alpha3 domain (73%) is comparable to those of others, suggesting domain-specific conservation of MR1. The localization of the mouse MR1 gene was determined to be chromosome 1H1, which corresponds to the human chromosomal region where the human MR1 gene is located (chromosome 1q25). High conservation of MR1 among mammals suggests that MR1 may be involved in critical conserved biological function(s).     

Characterization of a Schistosoma mansoni gene encoding a homologue of the Y-box binding protein

Hematopoietic stem cells (HSCs) support blood cells throughout life by utilizing their self-renewing and multilineage differentiating capabilities. Hematopoietic growth factors mediate their effects on stem cells by the tyrosine phosphorylation of proteins. Regulation of tyrosine phosphorylation is partially mediated by protein tyrosine phosphatases (PTPases). A possible mechanism by which hematopoietic stem cells maintain their self-renewing capacity and undifferentiated state is by controlling the balanced and opposing actions of protein tyrosine kinases (PTKs), receptors for growth factors, and PTPases. We have characterized the expression of PTPases in 5-fluorouracil (5-FU)-treated murine bone marrow cells, which represent a very primitive population of progenitors enriched for reconstituting stem cells, by using a consensus polymerase chain reaction (PCR) method. Several PTPases were expressed abundantly in the 5-FU-treated bone marrow stem cells. A novel PTP, termed protein tyrosine phosphatase receptor omicron (PTPRO), which is related to the homotypically adhering kappa, mu and PCP-2 receptor-type tyrosine phosphatases, was identified and characterized. We have cloned the murine and full-length human PTPRO cDNAs which share 89% homology, indicating that PTPRO is highly conserved between these species. The human PTPRO cDNA clone encodes a polypeptide of 1439 amino acids (aa) and has a calculated molecular mass of approximately 162 kDa. PTPRO consists of an extracellular segment containing a MAM domain, an immunoglobulin (Ig) domain, four fibronectin-type III (FN-III) repeats, a transmembrane segment, and two tandem intracellular PTP domains. The human PTPRO gene was assigned to human chromosome 1p35-pter using Southern blot analyses of genomic DNAs from rodent/human somatic hybrid cell lines containing human chromosome 1 or the p35-pter region of the chromosome. The mouse Ptpro gene was mapped to chromosome 4, closely linked to D4Mit16 and Elp1 (elliptocytosis-1), by using genomic DNAs from a (C57BL/6J x Mus spretus)F1 x Mus spretus backcross. In fetal tissues, PTPRO expression was observed in the brain and lung, whereas lower levels were observed in the kidney. In adult tissues, PTPRO was less restricted and was observed in the lung, heart, skeletal muscle, prostate, testis, and in various areas of the brain, indicating that PTPRO expression is developmentally regulated. Expression of PTPRO was also observed in human CD34+ bone marrow cells and 5-FU-treated murine primitive stem cells. These results suggest a potential role for PTPRO in stem cell adhesion and in mediating homophilic cell-cell interactions in other cell types.     

CASP, a novel, highly conserved alternative-splicing product of the CDP/cut/cux gene, lacks cut-repeat and homeo DNA-binding domains, and interacts with full-length CDP in vitro

Human CDP/cut and its murine counterpart, cux1/CDP are homeodomain repressor proteins in the family of Drosophila Cut. Northern blot analysis reveals complex alternative splicing, including forms too small to encode the full 1505 amino acid protein. We have characterized a CDP/cut alternatively spliced cDNA (CASP) of 3.4 kb. Human CASP, a predicted 678 amino acid polypeptide, shares 400 amino acids with CDP, but has an alternate N terminal exon of 20 aa, and the C-terminal 258 amino acids diverge from CDP/cut entirely. As the unique C-terminus of CASP lacks the three 'cut-repeats' and homeodomain of CDP/cut, we predict it does not bind DNA. Murine CASP, 96% similar to human, shares these features. Database searches identify homologs in chicken (86% identical to human CASP) and yeast (29% identical to human). Murine CASP mRNA is ubiquitous in mouse tissues and in tissue-culture cell lines. We generated a specific antiserum against the unique C-terminus of CASP, and used this reagent to demonstrate that CASP protein is expressed as an approx. 80 kDa protein in human and murine cells. Co-translation of in vitro-translated CDP and CASP mRNA, followed by immunoprecipitation with specific anti-CASP IgG, shows that CASP polypeptide can from a complex with CDP. Studies of the intron/exon structure of the murine cux/CDP/mCASP locus (> 100 kb) reveal that the unique 3' exons of CASP are interposed between cut-repeats 2 and 3 of the cux gene. We speculate that a primordial CASP-like gene captured a cut-repeat-homeobox gene to give rise to the eukaryotic Cut/CDP family of proteins.     

Cloning and characterization of the cDNA for the murine iduronate sulfatase gene

Iduronate sulfatase (IDS; EC 3.1.6.13) is a lysosomal enzyme that acts on sulfate groups on C-2 positions of iduronic acid residues of the mucopolysaccharides dermatan and heparan sulfate. A deficiency of this enzyme activity in man leads to Hunter syndrome (Mucopolysaccharidosis type II). We report here the cloning and sequence characterization of the murine iduronate sulfatase cDNA which encodes 564 amino acid residues. Within the coding region the murine gene is 84.9 and 84.5 identical to the human gene at the nucleotide and amino acid levels, respectively. The two regions containing the putative catalytic site are especially well conserved. Genetic mapping of the murine Ids cDNA in an interspecific backcross confirms an X chromosomal location between Fmr-1 and Gabra3.     

Magnesium lithospermate B ameliorates cephaloridine-induced renal injury

Emery-Dreifuss muscular dystrophy (EMD) is an X-linked recessive disorder associated with muscle wasting, contractures, and cardiomyopathy. The responsible emerin gene has recently been identified and found to encode a serine-rich protein similar to lamina-associated protein 2 (LAP2), although the disease mechanism remains obscure. In order to pursue the pathophysiology of this disorder, we report here the isolation and characterization of the complete mouse emerin gene. The emerin cDNA was isolated from murine strain BALB/c, and the emerin gene was isolated from strain 129. The 2.9-kb mouse emerin gene was completely sequenced and found to be composed of 6 exons and encode a protein 73% identical to that of the human protein. Key similarities with LAP2 were found to be conserved, including critical LAP2 phosphorylation sites. Examination of the murine promoter revealed three previously unrecognized cAMP response elements (CRE) conserved between human and mouse. While Northern analysis shows emerin to be widely expressed in the mouse, as it is in humans, these promoter elements may indicate cAMP responsiveness. These data provide the necessary elements to further investigate EMD in a murine system.     

Characterization of the mouse Myoc/Tigr gene

Mutations in the myocilin (MYOC), also known as Trabecular meshwork-Inducible Glucocorticoid Response (TIGR) gene can lead to juvenile open-angle glaucoma in human and may be responsible for at least 3% of primary open-angle glaucoma. To develop a mouse model of primary open angle glaucoma, and to get deeper insight into the mechanisms of the MYOC/TIGR gene regulation and function, we have isolated and characterized full size mouse Myoc/Tigr cDNA and genomic clones. The mouse and human MYOC/TIGR genes have the same exon-intron structure and contain 3 exons, although the mouse gene is 6 kb shorter than the human gene (10 kb versus 16 kb) due to differences in the length of introns. The MYOC/TIGR gene encodes a moderately conserved protein, which is 82% identical between human and mouse. The encoded protein is 14 amino acids shorter at the N-terminus in the mouse than in the human (490 versus 504 amino acids). Mouse and human MYOC/TIGR genes show a similar pattern of expression in adult ocular and nonocular tissues. The mouse Myoc/Tigr gene was mapped to Chromosome 1 at position 82.8 cM from the centromere. All residues, which were identified in the human MYOC/TIGR protein as critical for glaucoma development, are conserved in the mouse Myoc/Tigr.     

YAC contigs of the Rab1 and wobbler (wr) spinal muscular atrophy gene region on proximal mouse chromosome 11 and of the homologous region on human chromosome 2p

Despite rapid progress in the physical characterization of murine and human genomes, little molecular information is available on certain regions, e.g., proximal mouse chromosome 11 (Chr 11) and human chromosome 2p (Chr 2p). We have localized the wobbler spinal atrophy gene wr to proximal mouse Chr 11, tightly linked to Rab1, a gene coding for a small GTP-binding protein, and Glnsps1, an intronless pseudogene of the glutamine synthetase gene. We have now used these markers to construct a 1.3-Mb yeast artificial chromosome (YAC) contig of the Rab1 region on mouse Chr 11. Four YAC clones isolated from two independent YAC libraries were characterized by rare-cutting analysis, fluorescence in situ hybridization (FISH), and sequence-tagged site (STS) isolation and mapping. Rab1 and Glns-ps1 were found to be only 200 kb apart. A potential CpG island near a methylated NarI site and a trapped exon, ETG1.1, were found between these loci, and a new STS, AHY1.1, was found over 250 kb from Rab1. Two overlapping YACs were identified that contained a 150-kb region of human Chr 2p, comprising the RAB1 locus, AHY1.1, and the human homologue of ETG1.1, indicating a high degree of conservation of this region in the two species. We mapped AHY1.1 and thus human RAB1 on Chr 2p13.4-p14 using somatic cell hybrids and a radiation hybrid panel, thus extending a known region of conserved synteny between mouse Chr 11 and human Chr 2p. Recently, the gene LMGMD2B for a human recessive neuromuscular disease, limb girdle muscular dystrophy type 2B, has been mapped to 2p13-p16. The conservation between the mouse Rab1 and human RAB1 regions will be helpful in identifying candidate genes for the wobbler spinal muscular atrophy and in clarifying a possible relationship between wr and LMGMD2B.