ABSTRACT
New comparative genome hybridization technology on NotI-microarrays is presented (Karolinska Institute International Patent WO02/086163). The method is based on comparative genome hybridization of NotI-probes from tumor and normal genomic DNA with the principle of new DNA NotI-microarrays. Using this method 181 NotI linking loci from human chromosome 3 were analyzed in 200 malignant tumor samples from different organs: kidney, lung, breast, ovary, cervical, prostate. Most frequently (more than in 30%) aberrations--deletions, methylation,--were identified in NotI-sites located in MINT24, BHLHB2, RPL15, RARbeta1, ITGA9, RBSP3, VHL, ZIC4 genes, that suggests they probably are involved in cancer development. Methylation of these genomic loci was confirmed by methylation-specific PCR and bisulfite sequencing. The results demonstrate perspective of using this method to solve some oncogenomic problems.
Subject(s)
Chromosomes, Human, Pair 3/metabolism , Epigenesis, Genetic , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/biosynthesis , Neoplasms, Glandular and Epithelial/metabolism , Oligonucleotide Array Sequence Analysis/methods , Chromosomes, Human, Pair 3/genetics , Female , Humans , Male , Neoplasm Proteins/genetics , Neoplasms, Glandular and Epithelial/genetics , Organ Specificity , Quantitative Trait Loci/geneticsABSTRACT
A plasmid expression vector (pCEQ3), using temperature-regulated transcription from the p'R promoter of bacteriophage lambda, has been constructed. The vector is derived from pBR327 in which the EcoRI-ClaI fragment of plasmid DNA is replaced with a 2.2-kb DNA module cI857-pR-Q-p'R-qut-t'R, consisting of two regions of the lambda genome. The first region contains the repressor gene cI857 and promoter pR; the second one contains gene Q and the late promoter p'R. When the repressor protein, product of the cI857 gene, becomes temperature-inactivated, it allows the promoter pR to initiate the transcription of the Q gene. The product of the Q gene, in turn, acts as a positive regulator of transcription from promoter p'R. The promoter activity of pR is fully repressed at a low temperature (30 degrees C) and transcription from p'R is terminated in the absence of Q gene product, but the shift of temperature up to 37 degrees C is sufficient to make the transcription from the p'R promoter highly active. Foreign genes can be inserted into the single EcoRI site downstream from the p'R promoter. The resultant constructions express extremely high levels of the cloned gene product in Escherichia coli.
Subject(s)
Bacteriophage lambda/genetics , Gene Expression Regulation , Genetic Vectors , Plasmids , Cloning, Molecular , Escherichia coli/genetics , Lac Operon , Promoter Regions, Genetic , Temperature , Transcription, Genetic , beta-Lactamases/biosynthesisABSTRACT
Plasmid expression vector using the temperature-regulated promoter P'R of bacteriophage lambda is described. The vector carries a combination of two regions of lambda cI857indgenome, that contain: 1) gene cI and promoter PR, and 2) gene Q and promoter P'R. Transcription or gene Q is initiated at promoter PR, which is controlled by cI857 repressor. The Q gene product acts as a positive regulator of RNA synthesis from P'R. At 37 degrees C, sufficient amounts of protein Q are synthesised to initiate the expression of the cloned gene from P'R. Inactivation of Q gene (by elimination of a single NcoI site) results in the loss of P'R expression activity in the vector. E. coli beta-galactosidase gene (lacZ) and human leukocyte interferon alpha 2 gene (ifn alpha 2) were cloned into a single EcoRI cleavage site under the control of P'R. These constructs express high levels of beta-galactosidase and interferon alpha 2 in E. coli at 37 degrees C.
