Coding sequence, genomic organization, chromosomal localization, and expression pattern of the signalosome component Cops2: the mouse homologue of Drosophila alien

The Drosophila alien gene is highly homologous to the human thyroid receptor interacting protein, TRIP15/COPS2, which is a component of the recently identified signalosome protein complex. We identified the mouse homologue of Drosophila alien through homology searches of the EST database. We found that the mouse cDNA encodes a predicted 443-amino-acid protein, which migrates at approximately 50 kDa. The gene for the mouse alien homologue, named Cops2, includes 12 coding exons spanning approximately 30 kb of genomic DNA on the central portion of mouse chromosome 2. Mouse Cops2 is widely expressed in embryonic, fetal, and adult tissues beginning as early as E7.5. Mouse Cops2 cDNA hybridizes to two mRNA bands in all tissues at approximately 2.3 and approximately 4 kb, with an additional approximately 1.9-kb band in liver. Immunostaining of native and epitope tagged proteins localized the mouse Cops2 protein in both the cytoplasm and the nucleus, with larger amounts in the nucleus in some cells.     

Gene structure of human and mouse methylenetetrahydrofolate reductase (MTHFR)

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate for homocysteine remethylation to methionine. A human cDNA for MTHFR, 2.2 kb in length, has been expressed and shown to result in a catalytically active enzyme of approximately 70 kDa. Fifteen mutations have been identified in the MTHFR gene: 14 rare mutations associated with severe enzymatic deficiency and 1 common variant associated with a milder deficiency. The common polymorphism has been implicated in three multifactorial diseases: occlusive vascular disease, neural tube defects, and colon cancer. The human gene has been mapped to chromosomal region 1p36.3 while the mouse gene has been localized to distal Chromosome (Chr) 4. Here we report the isolation and characterization of the human and mouse genes for MTHFR. A human genomic clone (17 kb) was found to contain the entire cDNA sequence of 2.2 kb; there were 11 exons ranging in size from 102 bp to 432 bp. Intron sizes ranged from 250 bp to 1.5 kb with one exception of 4.2 kb. The mouse genomic clones (19 kb) start 7 kb 5′ exon 1 and extend to the end of the coding sequence. The mouse amino acid sequence is approximately 90% identical to the corresponding human sequence. The exon sizes, locations of intronic boundaries, and intron sizes are also quite similar between the two species. The availability of human genomic clones has been useful in designing primers for exon amplification and mutation detection. The mouse genomic clones will be helpful in designing constructs for gene targeting and generation of mouse models for MTHFR deficiency. Received: 28 January 1998 / Accepted: 9 April 1998     

Cloning and Characterization of a Mouse σ1 Receptor

Abstract: A cDNA clone (S2-1a) isolated from a mouse brain cDNA library, using a guinea pig σ₁ cDNA as probe, has high homology to the predicted protein sequence of the guinea pig (88%) and human (90%) σ₁ receptors. Northern analysis revealed a major mRNA of ∼1.8 kb in a wide range of mouse tissues, with highest levels in brain, liver, kidney, and thymus. Southern analysis and chromosomal mapping in the mouse suggested a single-copy gene in region A5-B2 of chromosome 4. Expression of the clone in MCF-7 and CHO cells led to a pronounced increase in (+)-[³H]pentazocine binding with a selectivity profile consistent with σ₁ receptors. In vitro translation yielded a protein of ∼28 kDa, as did transfection of a probe containing the hemagglutinin (HA) epitope (S2-1a.HA) into CHO cells, as determined by western analysis using an antibody directed against HA. (+)-[³H]-Pentazocine binding to immunopurified HA-tagged receptor demonstrated conclusively that S2-1a.HA encodes a high-affinity (+)-[³H]pentazocine binding site with characteristics of a murine σ₁ receptor. An antisense oligodeoxynucleotide designed from S2-1a potentiated opioid analgesia in vivo.     

A novel mouse kinesin of the UNC-104/KIF1 subfamily encoded by the Kif1b gene

Kinesin and kinesin-related proteins are microtubule-dependent motor proteins that transport organelles. We have cloned and sequenced a full-length 9924 bp mouse cDNA for a new kinesin of the UNC-104/KIF1 subfamily. Northern blot analysis of mouse RNAs detected high levels of a 10 kb mRNA in brain and eye, but lower levels in other tissues. Human RNA dot-blot analysis detected this mRNA in all tissues examined, although at different levels. The overall structure of the new kinesin (predicted size 204 kDa) was most similar to mouse KIF1A; however, 2.1 kb of the 5' portion of the cDNA were identical to the published sequence for KIF1B (Nangaku, M., Sato-Yoshitake, R., Okada, Y., Noda, Y., Takemura, R., Yamazaki, H., Hirokawa, N., 1994. KIF1B, a novel microtubule plus end-directed monomeric motor protein for transport of mitochondria. Cell 79, 1209-1220). We localized the Kif1b gene to the distal end of mouse Chromosome 4 by haplotype analysis of an interspecific backcross from The Jackson Laboratory. We had previously mapped the gene for the novel kinesin to the same location (Gong, T.-W.L., Burmeister, M., Lomax, M.I., 1996b. The novel gene D4Mille maps to mouse Chromosome 4 and human Chromosome 1p36. Mamm. Genome 7, 790-791). We conclude, therefore, that the Kif1b gene generates two major kinesin isoforms by alternative splicing. The shorter 7.8 kb mRNA encodes a 130 kDa kinesin, KIF1Bp130, whereas the 10 kb mRNA encodes a 204 kDa kinesin, KIF1Bp204. In addition, alternative splicing of two exons in the conserved region adjacent to the motor domain generates four different isoforms of each kinesin, leading to eight kinesin isoforms derived from the Kif1b gene.     

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     

Cloning and functional analysis of the Sry-related HMG box gene, Sox18

The Sox gene family (Sry like HMG box gene) is characterised by a conserved DNA sequence encoding a domain of approximately 80 amino acids which is responsible for sequence specific DNA binding. We initially published the identification and partial cDNA sequence of murine Sox18, a new member of this gene family, isolated from a cardiac cDNA library. This sequence allowed us to classify Sox18 into the F sub-group of Sox proteins, along with Sox7 and Sox17. Recently, we demonstrated that mutations in the Sox18 activation domain underlie cardiovascular and hair follicle defects in the mouse mutation, ragged (Ra) (Pennisi et al., 2000. Mutations in Sox18 underlie cardiovascular and hair follicle defecs in ragged mice. Nat. Genet. 24, 434-437). Ra homozygotes lack vibrissae and coat hairs, have generalised oedema and an accumulation of chyle in the peritoneum. Here we have investigated the genomic sequences encoding Sox18. Screening of a mouse genomic phage library identified four overlapping clones, we sequenced a 3.25 kb XbaI fragment that defined the entire coding region and approximately 1.5 kb of 5' flanking sequences. This identified (i) an additional 91 amino acids upstream of the previously designated methionine start codon in the original cDNA, and (ii) an intron encoded within the HMG box/DNA binding domain in exactly the same position as that found in the Sox5, -13 and -17 genes. The Sox18 gene encodes a protein of 468 aa. We present evidence that suggests HAF-2, the human HMG-box activating factor -2 protein, is the orthologue of murine Sox18. HAF-2 has been implicated in the regulation of the Human IgH enhancer in a B cell context. Random mutagenesis coupled with GAL4 hybrid analysis in the activation domain between amino acids 252 and 346, of Sox18, implicated the phosphorylation motif, SARS, and the region between amino acid residues 313 and 346 as critical components of Sox18 mediated transactivation. Finally, we examined the expression of Sox18 in multiple adult mouse tissues using RT-PCR. Low-moderate expression was observed in spleen, stomach, kidney, intestine, skeletal muscle and heart. Very abundant expression was detected in lung tissue.     

栽培因子对胡麻天亚9号产量的影响

为了探讨甘肃省胡麻高产栽培方案,以‘天亚9号’为试验材料,采用播种量（X₁）、底施氮肥（X₂）、底施磷肥（X₃）、底施钾肥（X₄）、叶面施钾肥（X₅）、叶面施硼肥（X₆）、生长调节剂（多效唑,X₇）、生育期灌水量（X₈）的8因素均匀设计,研究不同栽培因子对高产优质胡麻‘天亚9号’籽粒产量的影响,并对胡麻产量与各栽培因子进行相关性分析、通径分析及主成分分析.结果表明： 影响胡麻产量的因素为播量、底施氮肥、底施钾肥、生长调节剂、底施磷肥、叶面喷施钾肥;其相关性依次为播量>生长调节剂（多效唑）>底施氮肥>底施磷肥>叶面喷施钾肥>底施钾肥.进一步进行最高产量模拟寻优,采用频数分析法得到胡麻产量大于173.58 kg·hm^-2的优化栽培因子为：播量4.68～4.92 kg·hm^-2,底施氮肥11.59～14.75 kg·hm^-2,底施磷肥17.26～21.95 kg·hm^-2,底施钾肥7.00～12.50 kg·hm^-2,叶面肥（磷酸二氢钾）1.41～1.81 kg·hm^-2,生长调节剂（多效唑）751.74～954.04 g·hm^-2.