Differential Display Reverse Transcription Polymerase Research Articles

Use of differential display reverse transcription – polymerase chain reaction for discovery of induced adrenomedullin gene expression in focal stroke

Differential display and other techniques such as subtractive library screening and representational difference analysis have been developed and widely used for novel gene discovery in pathological and specific physiological conditions. In the present work, we illustrate the utilization of the differential display method for the investigation of the differentially expressed genes in a rat model of focal brain ischemia. Adrenomedullin, a recently discovered vasodilator peptide, was identified to be upregulated after focal brain ischemia and further demonstrated to play a pathophysiological role in exacerbating ischemic damage. The data suggest a high potential for the application of mRNA differential display, as well as other techniques, for the discovery of novel therapeutic targets.

Expression of a novel mRNA in human head and neck squamous cell carcinoma cells.

AIMS: The differential display reverse transcription polymerase chain reaction (DDRT-PCR) technique was used to search for differences between the mRNA expression profiles of squamous cell carcinoma (SCC) cell lines established...

Complexity of IL-1β induced gene expression pattern in human articular chondrocytes

The mRNA fingerprinting technique, differential display reverse transcription polymerase chain (DDRT-PCR), was used to detect changes in the overall pattern of gene expression in human articular knee chondrocytes-induced by interleukin-1 beta (IL-1 beta), the prototypical inducer of catabolic responses in degenerate joint diseases. One hundred different primer combinations generated approximately 10,000 different PCR fragments for IL-1 beta treated, as well as for untreated human chondrocytes, cultivated in alginate beads. This represented 53% of all expressed chondrocyte genes as based on statistical considerations. Side by side comparisons of differential display patterns originating from two different donor tissues yielded 44 reproducibly, differentially-displayed cDNA fragments, which were subcloned and sequenced. Sequence homology searches revealed sequence identities to the human necrosis factor alpha (TNF-alpha) and IL-1 regulated gene TSG-6, fibronectin, osteopontin, calnexin, and the DNA repair enzyme ERCC5. The differential expression was confirmed with Northern and quantitative PCR analyses. The known function of these genes and their known IL-1 responsiveness indicate that the employed model system reflects the pleiotropic effects of IL-1 on the overall gene expression in human articular chondrocytes and identifies genes involved in very different biochemical pathways. Twenty-seven cDNAs lacked sequence homologies to known genes and may represent novel genes.

CDNAs encoding the antigenic proteins in pathogenic strain of Entamoeba histolytica

The differential display reverse transcription polymerase chain reaction (DDRT-PCR) analysis was performed to identify the pathogenic strain specific amplicons. mRNAs were purified from the trophozoites of the pathogenic strain YS-27 and the non-pathogenic strain S 16, respectively. Three kinds of first stranded cDNAs were reverse transcribed from the mRNAs by one base anchored oligo-dT11M (M: A, C, or G) primers. Each cDNA template was used for DDRT-PCR analysis. A total of 144 pathogenic strain specific amplicons was observed in DDRT-PCR analysis using primer combinations of the 11 arbitrary primers and the 3 one base anchored oligo-dT11M primers. Of these, 31 amplicons were verified as the amplicons amplified only from the mRNAs of the pathogenic strain by DNA slot blot hybridization. Further characterization of the 31 pathogenic strain specific amplicons by DNA slot blot hybridization analysis using biotin labeled probes of the PCR amplified DNA of cysteine proteinase genes revealed that 21 of them were amplified from the mRNAs of the cysteine proteinase genes. Four randomly selected amplicons out of the rest 10 amplicons were used for screening of cDNA library followed by immunoscreening and all of them were turned out to be amplified from the mRNA.

Isolation and characterization of cDNA for a plant mitochondrial phosphate translocator (Mpt1): ozone stress induces Mpt1 mRNA accumulation in birch (Betula pendula Roth).

We have isolated by DDRT-PCR (differential-display reverse-transcription polymerase chain reaction) and cDNA library screening a 1.3 kb cDNA corresponding to a strongly ozone-inducible transcript from birch (Betula pendula Roth). Nucleotide sequence analysis suggests that it encodes a mitochondrial phosphate translocator protein (Pic), the first one isolated from plants. The isolated birch mitochondrial phosphate translocator cDNA (designated Mpt1) contains an open reading frame of 1092 bases encoding a 364 amino acid polypeptide. The deduced protein is 66% similar to bovine Pic isoform B. Comparison of the N-terminal amino acid sequence to known mammalian Pic proteins and the existence of an in-frame stop codon upstream of the initiation codon suggest that the isolated cDNA is full-length. Southern hybridization analysis of birch genomic DNA shows that Mpt1 is a single-copy gene. Accumulation of Mpt1 mRNA during oxidative stress imposed by ozone is detectable already at 2 h and it is at maximum ca. 12 h after the beginning of an 8 h ozone exposure (150 ppb). A second O3 peak at 48-56 h did not increase transcript levels further. O3 exposure for 2 h was sufficient for Mpt1 induction. Birch Mpt1 transcript levels remain at moderately low level during leaf development and is lower in roots and leaves when compared to young shoots undergoing wood formation and lignification.

The Human Homologue of the Yeast CHL1 Gene Is a Novel Keratinocyte Growth Factor-regulated Gene

Keratinocyte growth factor (KGF) is a potent and specific mitogen for different types of epithelial cells, including keratinocytes of the skin. To gain insight into the mechanisms of KGF action in this tissue, we attempted to identify genes that are regulated by KGF in keratinocytes. Using the differential display reverse transcription polymerase chain reaction technology, a gene was identified which was strongly induced in these cells by treatment with KGF but not with serum growth factors or pro-inflammatory cytokines. This gene seems to be part of a multigene family as assessed by Southern blot analysis. Molecular cloning and sequencing of the full-length cDNA revealed a strong homology with the yeast CHL1 gene. The latter encodes a putative helicase, which is involved in correct chromosome transmission and cell cycle progression. Furthermore, the CHL1 gene product and the protein encoded by the novel KGF-regulated gene were identical in size, indicating that we had cloned the human CHL1 homologue. This finding suggests a novel and specific role of KGF in correct chromosome segregation and/or cell cycle progression.

Arbitrary primed RNA fingerprinting in plants

The technique of differential display reverse transcription polymerase chain reaction (DDRT-PCR) was used to identify different m-RNA species between two unique populations of Brassica napus L. Numerous polymorphic bands were observed between untreated and methyl jasmonate-treated tissue. This method has the potential to identify induced, differentially expressed genes in plants.