ABSTRACT
Ubiquitin is a critical modifier regulating the degradation and function of its target proteins during posttranslational modification. Here we found that ubiquitin-specific peptidase 24 (USP24) is highly expressed in cell lines with enhanced malignancy and in late-stage lung cancer clinical samples. Studying single-nucleotide polymorphisms (SNPs) of USP24 using genomic DNA of lung cancer patients revealed an increase in SNP 7656C/T. When using RNA specimens instead of the genomic DNA of lung cancer patients, we found significant increases in the ratios of variants 930C/T and 7656T/C, suggesting that variants at these two sites are not only caused by the SNP of DNA but also by the RNA editing. USP24-930T and USP24-7656C increase USP24 expression levels by increasing RNA stability. Knocking down USP24 increased Suv39h1 level through a decrease in mouse double-minute 2 homolog levels, thus enhancing lysine-9 methylation of histone H3, and resulting in the prevention of lung cancer malignancy. In conclusion, as USP24 variant analysis revealed a higher ratio of variants in blood specimens of lung cancer patients than that in normal individuals, USP24-930T and USP24-7656C might be useful as diagnostic markers for cancer detection.
Subject(s)
Genetic Predisposition to Disease/genetics , Lung Neoplasms/genetics , Polymorphism, Single Nucleotide , Ubiquitin Thiolesterase/genetics , A549 Cells , Animals , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Blotting, Western , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Genotype , Humans , Immunohistochemistry , Lung Neoplasms/blood , Lung Neoplasms/metabolism , Male , Methyltransferases/genetics , Methyltransferases/metabolism , Mice, Inbred BALB C , Mice, Nude , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , RNA Interference , RNA Stability/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Survival Analysis , Transplantation, Heterologous , Ubiquitin Thiolesterase/metabolismABSTRACT
A 2.1-kilobase fragment obtained by restriction enzyme HindIII digestion of Erwinia sp. genomic DNA was cloned into plasmid pUC19 and introduced into Escherichia coli by transformation. The transformants with diphenyl ether cleaving activity (Dpe+) were selected on agar plates with a specially designed medium (LTFN) containing 4-nitrodiphenyl ether. The positive clones showed a clear zone around the colonies. Analysis of mutants obtained by transposon mini-Mu dI(lacZ Kmr) mutagenesis indicated the coding region of the gene (dpe) and the utilization of a lacZ promoter of pUC19 for transcription of dpe. Clones with dpe in the opposite orientation in pUC19 were not expressed, confirming the need for a lacZ promoter. Utilization of a lacZ promoter in pUC19 was further confirmed by the observation that the degradation of 4-nitrodiphenyl ether was enhanced in the presence of isopropyl-beta-D-thiogalactoside. Expression of dpe was also found in pDPE7321, generated from cloning this gene into another plasmid, pSP73. Analysis of the plasmid-encoded proteins by the maxicell technique showed a polypeptide of 21,000 molecular weight as the product of dpe.
Subject(s)
Erwinia/genetics , Escherichia coli/genetics , Genes, Bacterial , Phenyl Ethers/metabolism , Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Biodegradation, Environmental , Chromatography, High Pressure Liquid , Cloning, Molecular , DNA, Bacterial/isolation & purification , Escherichia coli/metabolism , Genetic Vectors , Plasmids , Restriction Mapping , Sequence Homology, Nucleic AcidABSTRACT
An Acinetobacter strain PE7 with the ability to grow on salicyclic acid and to degrade diphenyl ethers was isolated from a petroleum waste pit in Louisiana. A cloned Erwinia sp. dpe gene encoding diphenyl ether cleavage was introduced into PE7 in order to enhance its degradative ability. A broad-host-range expression plasmid, pDPE2388, was constructed by inserting an SspI-HpaI fragment from a dpe gene-containing plasmid, pDPE7321, into the kanamycin resistance gene of plasmid pKT230. The DNA fragment contained the dpe gene flanked between sp6 and T7 promoters. Transconjugants of pDPE2388 plasmid into PE7 were isolated. Expression of the dpe gene in Escherichia coli or PE7 displayed a degradative ability to cleave the following diphenyl ethers: 4-chlorodiphenyl ether, 4-nitrodiphenyl ether, and 4-hydroxydiphenyl ether.
Subject(s)
Acinetobacter/metabolism , Erwinia/metabolism , Escherichia coli/metabolism , Genes, Bacterial , Phenyl Ethers/metabolism , Acinetobacter/genetics , Acinetobacter/isolation & purification , Biodegradation, Environmental , Conjugation, Genetic , Erwinia/genetics , Gene Expression Regulation, Bacterial , Industrial Waste , Petroleum , Plasmids , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Salicylates/metabolism , Salicylic AcidABSTRACT
Two halophilic anaerobic bacteria, one of which had chitinolytic activity, were isolated from a solar saltern in southern California. These organisms were long, gram-negative, motile, flexible rods. The biochemical and physiological characteristics of these bacteria were very similar but were different from the characteristics of other haloanaerobic bacteria. Both grew at salt concentrations ranging from 0.5 to 5 M and at temperatures ranging from 23 to 50 degrees C. They were sensitive to chloramphenicol but resistant to penicillin, carbenicillin, d-cycloserine, streptomycin, and tetracycline. An analysis of DNAs and whole-cell proteins showed that they were closely related taxonomically and distinguishable from other halophilic anaerobic bacteria. They exhibited 92.3 to 100% DNA homology as determined by DNA-DNA hybridization. The guanine-plus-cytosine contents of their DNAs were 34.8+/-1 mol%. The two isolates, strains W5C8 and W3C1, differed from other halophilic anaerobic bacteria sufficiently to support establishment of a new genus and species, Haloanaerobacter chitinovorans. Strain W5C8 exhibited chitinolytic activity and is designated the type strain. Two chitin-induced extracellular proteins with molecular weights of 38 x 10 and 40 x 10 were detected in strain W5C8.