ABSTRACT
Kainic acid (KA) causes neurodegeneration, but no consensus has been reached concerning its mechanism. Nitric oxide may be a regulator of the mechanism. We identified differentially expressed genes in the hippocampus of mice treated with kainic acid, together with or without L-NAME, a nonselective nitric oxide synthase inhibitor, using a new differential display PCR method based on annealing control primers. Eight genes were identified, including clathrin light polypeptide, TATA element modulatory factor 1, neurexin III, ND4, ATPase, H+ transporting, V1 subunit E isoform 1, and N-myc downstream regulated gene 2. Although the functions of these genes and their products remain to be determined, their identification provides insight into the molecular mechanism(s) involved in KA-induced neuronal cell death in the hippocampal CA3 area.
Subject(s)
Animals , Mice , Cell Death , Clathrin , Consensus , Hippocampus , Kainic Acid , Models, Animal , Neurons , NG-Nitroarginine Methyl Ester , Nitric Oxide Synthase , Nitric Oxide , Polymerase Chain Reaction , Proton-Translocating ATPasesABSTRACT
Differential display is a widely used methodology to identify genes that are differentially expressed in biological samples. We developed a new protocol for the amplification and recovery of differentially expressed genes from extremely small initial amounts of RNA (10 to 25 pg mRNA) from preimplantation bovine embryos. The cDNAs generated with an anchor primer, associated with a universal sequence, were amplified with an arbitrary primer and a single fluorescently labeled primer. Amplification products were easily visualized with a fluorescence scanner, without the need for radioisotopes. Nineteen isolated fragments were cloned and sequenced, confirming the expected primer sequences and allowing the recognition and identification of gene transcripts involved in bovine embryonic physiology.
Subject(s)
Animals , Cattle , Blastocyst , Embryonic Development/genetics , Fertilization in Vitro/methods , Polymerase Chain Reaction/methods , DNA, Complementary/genetics , Molecular Sequence Data , Expressed Sequence Tags , Fluorescence , RNA, Messenger/genetics , Gene Expression Regulation, Developmental , Base Sequence , Transcription, GeneticABSTRACT
OBJECTIVES: This study was performed to identify genes regulated by electroconvulsive shock (ECS) and to observe the pattern of expression of genes according to different developmental stages and brain regions. METHODS: ECS(130V, 0.5 sec) was given to male Sprague-Dawley rats with age of postnatal day 7 and 21(P7, P21 respectively). After screening genes regulated by ECS with mRNA differential display-PCR(DD-PCR), we selected one clone among them and observed the induction of this gene after ECS by time-dependent Northern blot analysis of rat brain of P7, P21 and adult rat cortex and hippocampus. RESULTS: By DD-PCR method, we have identified four clones whose expression was regulated by ECS. Among them, one(CP 10-2) was proved to be a new gene by sequencing and BLAST search. Its expression was increased after ECS in P7, P21, and adult rat brain. The expression of CP 10-2 reached peak level at 180 minutes after ECS in P7 rat brain, but was further increased until 360 minutes after ECS in P21 and adult rat brain. CONCLUSION: In this study, a new gene was identified in rat brain which showed up-regulated expression in response to ECS. Cloning and characterization of this new gene would be helpful to elucidate the effect of ECS in rat brain.
Subject(s)
Adult , Animals , Humans , Male , Rats , Blotting, Northern , Brain , Clone Cells , Cloning, Organism , Electroshock , Hippocampus , Mass Screening , Rats, Sprague-Dawley , RNA, MessengerABSTRACT
PURPOSE: This study was aimed to isolate cDNAs putatively associated with doxorubicin resistance or sensitivity in gastric carcinoma cell line. MATERIALS AND METHODS: A doxorubicin-sensitive parental SNU-16 and doxorubicin resistant SNU-16DOX cell line were used. Differential display-PCR(DD-PCR) was employed to screen for differentially expressed cDNA fragment either in parental or resistant cell line and followed by subtractive hybridization to discriminate true positive from false positive clones. The sequences were determined and compared to the sequence data base registered at the GenBank. RESULTS: Four clones(16, 19, 21, and 22 clone) were isolated, of which three(16, 19 and 21 clone) was downexpressed, and one(22 clone) was overexpressed in doxorubicin resistant cell line. All four clones were found to be novel sequences. Further analysis for these clones are under characterization. CONCLUSION: Four partial cDNA clones that are putatively associated with doxorubicin resistance or sensitivity in gastric carcinoma cell line were isolated.
Subject(s)
Humans , Cell Line , Clone Cells , Databases, Nucleic Acid , DNA, Complementary , Doxorubicin , ParentsABSTRACT
Differential display-PCR was used to clone the differential expressed genes between Mycobacterium tuberculosis virulence strain H37Rv and its avirulent mutant H37Ra. All of different genes were cloned, sequenced and some were analyzed by Northern-blotting. Two cDNAs that appeared to be expressed in H37Rv, but not in H37Ra, were cloned and sequenced. Rv0170, and Rv1894c, code for proteins with unknown functions. The two gene were present in H37Ra, but not expressed. These results show that mRNA DD methodology can represent a potential tool for investigation of M. tuberculosis gene expression.