ABSTRACT
The search for differentially expressed genes in gastric cancer may help define molecular alterations and molecular diagnosis of gastric cancer. Using the differential display PCR technique, we identified 18 genes that are differentially expressed between normal and tumor human gastric tissues. Their expressions were verified with reverse Northern blot analysis and Northern blot analysis. Oxidative phosphorylation-related genes, antizyme inhibitor of ornithine decarboxylase, protein phosphatase-1beta, 35-kDa peroxisomal membrane protein, and cystic fibrosis transmembrane conductance receptor were highly expressed in tumor tissue, whereas pepsinogen A, Na-K ATPase alpha subunit, nerve growth factor receptor, and alpha-tropomyosin were highly expressed in normal tissue. In addition, 3 unknown genes were found to be differentially expressed in paired gastric tissues. These differentially expressed genes may provide significant opportunities for further understanding of gastric carcinogenesis and the molecular diagnosis of gastric cancer.
Subject(s)
Gastric Mucosa/metabolism , Gene Expression , Stomach Neoplasms/genetics , DNA, Complementary , DNA, Neoplasm/analysis , Enzyme Inhibitors , Gene Expression Profiling/methods , Humans , Polymerase Chain Reaction/methods , Sequence Analysis, DNA/methods , Stomach Neoplasms/pathologyABSTRACT
To elucidate the role of the -35 sequence and its cooperativity with vir box in the expression of the virE gene, various mutants were constructed by either site-directed mutation or deletional mutation of the virE promoter. The expression level of pHBAV, a mutant where its putative -35 sequences (CCGAGT) have been substituted with the consensus -35 sequences of the Escherichia coli promoter (TTGACA), was increased by 386%. pECHV, containing the conserved -35 sequence but lacking the vir box and the 5'-half of the imperfect dyad symmetry region (DSR) showed an increase of 286% in its promoter activity. pESHV, containing the conserved -35 sequence but lacking the complete 5'-upstream region from the mid-region of imperfect DSR, exhibited 244% of the native virE promoter activity. pHBCA, containing the conserved -35 sequence but destroying the vir box, was constructed by substitution of A, C, T at the positions -62, -63, and -65 on the vir-box to T, A, C, respectively. These mutations increased promoter activities by 319%. On the other hand, when the vir box was mutated from imperfect DSR to almost perfect DSR with T to A and G to T substitutions at -60 and -61 positions of the virE promoter containing the conserved -35 sequence (pHBNA), a higher activity of 671% was observed. These results demonstrate that when the putative -35 sequence of virE promoter is replaced with the consensus -35 sequence, the virE gene can be expressed independently without the binding of VirG protein to the vir-box and/or the induction of acetosyringone. Moreover, the presence of an almost perfect dyad symmetry of the vir-box can increase the expression of virE synergistically with the consensus -35 sequence.
Subject(s)
Agrobacterium tumefaciens/genetics , Bacterial Proteins/genetics , DNA, Bacterial/genetics , Gene Expression Regulation, Bacterial , Regulatory Sequences, Nucleic Acid , Virulence Factors , Agrobacterium tumefaciens/metabolism , Bacterial Proteins/biosynthesis , Base Sequence , Consensus Sequence , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Molecular Chaperones/biosynthesis , Molecular Chaperones/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Sequence DeletionABSTRACT
Agrobacterium tumefaciens pTiA6 virE promoter has a vir box, an inverted repeat (IR) sequence, a putative -35 region and a consensus -10 region. To study how the IR sequence of the virE promoter plays a role in virE gene expression, various mutants were constructed by base substitution and deletion in the virE promoter region. Substitution of the 3'-end region of the IR sequence, 5'-TCCGTTTCAA-3' to 5'-GCGGCCGCTC-3' displayed 2.6% of the native virE promoter activity. A deletion mutant of 5'-CGTTTCAA-3' on the 3'-end region of the IR sequence expressed 6% of the native virE promoter activity. These mutational analyses demonstrated that the IR sequence of the virE promoter plays a role as a cis-acting element in virE expression.
