FIGC, a novel FGF-induced ubiquitin-protein ligase in gastric cancers

We have previously shown that fibroblast growth factor receptor 2 (FGFR2) plays an important role in gastric carcinogenesis. In this study, we have used a differential display approach to identify basic fibroblast growth factor (bFGF)-inducible genes in gastric cancer cells. Here, we report that one of these genes is predicted to encode a RING finger protein, designated FIGC. The FIGC gene was found to encode a polypeptide of 381 amino acids with a novel RING finger module at the NH2-terminus and the COOH-terminal proline-rich region. Using an in vitro ubiquitination assay with recombinant protein, we demonstrate that FIGC has intrinsic E3 ubiquitin ligase activity and promotes ubiquitination. Our data indicate that FIGC upregulation in response to bFGF in gastric cancer might be implicated in carcinogenesis through dysregulation of growth modulator.     

LITAF、RAB7、LMNA和MTMR2基因在中国人腓骨肌萎缩症患者的突变分析

为了分析LITAF、RAB7、LMNA和MTMR2基因在中国人腓骨肌萎缩症(Charcot-Marie-Tooth disease, CMT)的突变特点, 文章分别应用PCR结合DNA序列分析方法和PCR-单链构象多态性(PCR-SSCP)结合DNA序列分析方法对6个常染色体显性遗传家系先证者和27个散发病例进行LITAF和RAB7基因突变分析; 应用PCR-SSCP结合DNA序列分析方法对14个常染色体遗传的CMT家系先证者和27个散发患者进行LMNA和MTMR2基因突变分析。结果发现: LITAF基因c.269G→A、c.274A→G序列变异和LMNA基因c.1243G→A、c.1910C→T序列变异, 未发现RAB7和MTMR2基因的序列变异。其中LITAF基因c.269G→A、LMNA基因c.1243G→A和c.1910C→T为新发现的单核苷酸多态; LITAF基因c.274A→G为已知多态。说明LITAF、RAB7、LMNA和MTMR2基因突变在中国人CMT患者中罕见。     

Isolation and characterization of a novel RING-H2 finger gene induced in response to cold and drought in the interfertile Citrus relative Poncirus trifoliata

Many genes in different organisms encode proteins with really interesting gene (RING) finger domain(s). The RING zinc finger domain is involved in a wide variety of functions in diverse organisms. A cDNA clone showing homology with RING zinc finger genes and nine-fold induction in response to cold was previously identified during a gene expression study in the interfertile Citrus relative Poncirus trifoliata (L.) Raf. In this study, the full-length cDNA of this clone was isolated from 2-day cold-acclimated P. trifoliata by a rapid amplification of cDNA ends method using gene-specific primers. The full-length cDNA was 956 bp containing a complete open reading frame of 474 bp encoding a polypeptide of 158 amino acids. The full-length cDNA showed a high level of homology with genes encoding putative RING zinc finger proteins in plants. The deduced amino acid sequence of this gene contained a signature sequence motif for a RING zinc finger close to the C terminus of the protein. The RING zinc finger domain was significantly similar to previously characterized RING zinc finger proteins from different organisms. Additionally, it had a histidine residue at the fifth co-ordination site, indicating that this gene encodes a RING-H2 finger protein. Northern blot hybridization showed that the expression of the RING finger gene was induced in response to cold in cold-hardy P. trifoliata but not to the same extent in cold-sensitive Citrus grandis L. Osb. (pummelo). However, the gene was induced by drought stress similarly in both the species. To our knowledge, this study presents the first isolation of the full-length sequence of a RING zinc finger gene induced in response to abiotic stress in plants and the initial characterization of this gene in Citrus .     

PIG7慢病毒表达载体的构建

目的:构建一个含有PIG7基因ORF区的表达载体pPRRL-PIG7-IRES,为研究PIG7基因的功能打下基础。方法:采用RT-PCR方法从经苯丁酸钠处理过的Kasumi-1细胞获得PIG7基因SIMPLE转录本的ORF片段,再用酶切-连接的方法将目的片段亚克隆入慢病毒表达质粒PRRL.SIN.CPPT.PGK/GFP.WPRE。结果:PIG7基因ORF区成功亚克隆入了慢病毒表达质粒PRRL.SIN.CPPT.PGK/GFP.WPRE。结论:成功完成了PIG7慢病毒表达载体的构建,为研究PIG7基因的功能打下了基础。     

Cloning of a catabolite repression control (crc) gene from Pseudomonas aeruginosa, expression of the gene in Escherichia coli, and identification of the gene product in Pseudomonas aeruginosa.

Mutants which are defective in catabolite repression control (CRC) of multiple independently regulated catabolic pathways have been previously described. The mutations were mapped at 11 min on the Pseudomonas aeruginosa chromosome and designated crc. This report describes the cloning of a gene which restores normal CRC to these Crc- mutants in trans. The gene expressing this CRC activity was subcloned on a 2-kb piece of DNA. When this 2-kb fragment was placed in a plasmid behind a phage T7 promoter and transcribed by T7 RNA polymerase, a soluble protein with a molecular weight (MW) of about 30,000 was produced in Escherichia coli. A soluble protein of identical size was overproduced in a Crc- mutant when it contained the 2-kb fragment on a multicopy plasmid. This protein could not be detected in the mutant containing the vector without the 2-kb insert or with no plasmid. When a 0.3-kb AccI fragment was removed from the crc gene and replaced with a kanamycin resistance cassette, the interrupted crc gene no longer restored CRC to the mutant, and the mutant containing the interrupted gene no longer overproduced the 30,000-MW protein. Pools of intracellular cyclic AMP and the activities of adenylate cyclase and phosphodiesterase were measured in mutant and wild-type strains with and without a plasmid containing the crc gene. No consistent differences between any strains were found in any case. These results provide original evidence for a 30,000-MW protein encoded by crc+ that is required for wild-type CRC in P. aeruginosa and confirms earlier reports that the mode of CRC is cyclic AMP independent in this bacterium.     

Characterization, chromosomal localization, and expression during hematopoietic differentiation of the gene encoding Arl6ip, ADP-ribosylation-like factor-6 interacting protein (ARL6)

Proliferation and differentiation of hematopoietic stem cells and progenitors are regulated by signals from the microenvironment, involving both secreted cytokines and adhesion molecules. The exact mechanisms by which cytokines act on hematopoietic development are still not well understood. To extend the molecular characterization of gene regulation during cytokine-induced hematopoiesis, we applied mRNA differential display to identify genes regulated when multipotent progenitor cells are allowed to differentiate into monocytes and neutrophils. Here we report the isolation and characterization of a gene that is downregulated during myeloid differentiation and encodes a 23-kDa protein with four putative transmembrane segments. The gene, which we named Arl6ip, is identical to a mouse gene recently identified by its physical interaction with ADP-ribosylation-like factor-6 (ARL6), belonging to the Ras superfamily. We add information on its full-length characterization as well as its regulation during hematopoiesis. It is expressed in all hematopoietic cell lineages, but the highest level of expression is found in early myeloid progenitor cells. Preliminary studies by immunofluorescence microscopy revealed that the ARL6IP protein is predominantly localized to intracytoplasmic membranes. This suggests an involvement of the Arl6ip gene in protein transport, membrane trafficking, or cell signaling during hematopoietic maturation.     

Plasmid location,cloning, and sequence analysis of the gene encoding a 27.3-kilodalton cytolytic protein fromBacillus thuringiensis subsp.morrisoni (PG-14)

A gene encoding the 27.3 kilodalton cytolytic protein toxin in the mosquitocidal isolate (PG-14) ofBacillus thuringiensis subsp.morrisoni (BTM) was cloned and sequenced, and compared with the homologous gene inB. thuringiensis subsp.israelensis (BTI). The BTM gene was determined to be located on a 140 kb plasmid by use of a synthetic 20-base nucleotide probe derived from the sequence of the BTI gene. A 9.4-kbHind III plasmid fragment containing the BTM cytolytic protein gene was cloned into the plasmid vector pUC12, and subsequently subcloned and sequenced. Comparison of the nucleotide sequence of the BTM gene with that of the homologous BTI gene revealed only a single base difference; the base at position 310 in BTM is guanine, whereas in BTI it is cytosine. This single base change results in the occurrence of alanine rather than proline at amino acid residue 82 in BTM. Analysis of the secondary structure and hydropathic profile of the BTM protein indicates that alanine at this position increases both the propensity to form an -helical structure and the hydrophobicity in the vicinity of this residue. Thus, the BTM toxin is potentially more cytolytic than the homologous protein of BTI.     

A gene family encoding RING finger proteins in rice: their expansion, expression diversity, and co-expressed genes

The proteins harboring RING finger motif(s) have been shown to mediate protein–protein interactions that are relevant to a variety of cellular processes. In an effort to elucidate the evolutionary dynamics of the rice RING finger protein family, we have attempted to determine their genomic locations, expression diversity, and co-expressed genes via in silico analysis and semi-quantitative RT–PCR. A total of 425 retrieved genes appear to be distributed over all 12 of the chromosomes of rice with different distributions, and are reflective of the evolutionary dynamics of the rice genome. A genome-wide dataset harboring 155 gene expression omnibus sample plates evidenced some degree of differential evolutionary fates between members of RING-H2 and RING-HC types. Additionally, responses to abiotic stresses, such as salinity and drought, demonstrated that some degree of expression diversity existed between members of the RING finger protein genes. Interestingly, we determined that one RING-H2 finger protein gene (Os04g51400) manifested striking differences in expression patterns in response to abiotic stresses between leaf and culm-node tissues, further revealing responses highly similar to the majority of randomly selected co-expressed genes. The gene network of genes co-expressed with Os04g51400 may suggest some role in the salt response of the gene. These findings may shed further light on the evolutionary dynamics and molecular functional diversity of these proteins in complex cellular regulations.     

Regulation of the interleukin-1 beta (IL-1 beta) gene by mycobacterial components and lipopolysaccharide is mediated by two nuclear factor-IL6 motifs.

The cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) are released by mononuclear phagocytes in vitro after stimulation with mycobacteria and are considered to mediate pathophysiologic events, including granuloma formation and systemic symptoms. We demonstrated that the Mycobacterium tuberculosis cell wall component lipoarabinomannan (LAM) is a very potent inducer of IL-1 beta gene expression in human monocytes and investigated the mechanism of this effect. We localized the LAM-, lipopolysaccharide (LPS)-, and TNF-alpha-inducible promoter activity to a -131/+15 (positions -131 to +15) DNA fragment of the IL-1 beta gene by deletion analysis and chloramphenicol acetyltransferase assay. Within this DNA fragment, there were two novel 9-bp motifs (-90/-82 and -40/-32) with high homology to the nuclear factor-IL6 (NF-IL6) binding site. Site-directed mutagenesis demonstrated that the two NF-IL-6 motifs could be independently activated by LAM, LPS, or TNF-alpha and that they acted in an orientation-independent manner. DNA mobility shift assay revealed specific binding of nuclear protein(s) from LAM-, LPS-, or TNF-alpha-stimulated THP-1 cells to the NF-IL6 motifs. We conclude that the two NF-IL6 sites mediate induction of IL-1 beta in response to the stimuli LAM, LPS, and TNF-alpha.