Polymerase reaction without primers throughout for the reconstruction of full-length cDNA from products of rapid amplification of cDNA ends (RACE)
Department of Respiratory Medicine, University of Tokyo Hospital, Tokyo, Japan. Biotechnology Letters
(Impact Factor: 1.59).
03/2011; 33(7):1301-7. DOI: 10.1007/s10529-011-0580-1
Rapid amplification of cDNA ends (RACE) has widely been used to determine both ends of the cDNA from its partial sequence. Conventionally, 5'- and 3'-RACE products were ligated at a restriction site in the overlap region to reconstruct the full-length cDNA; however, reconstruction is difficult if no appropriate restriction enzymes are available. Here, we report a novel method to reconstruct full-length cDNA with DNA polymerase. Instead of usual PCR, chain reactions were avoided and the elongation time was shortened, which enables non-specific products or undesired point mutations to be minimized. We successfully reconstructed and TA-cloned a full-length cDNA of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene variant 2 from RACE products obtained from a surgically resected lung adenocarcinoma sample. We also evaluated some parameters to provide recommendations for this new method.
Available from: europepmc.org
- "To construct the FER expression plasmid vector , full-length human FER was cloned using a previously reported method . Briefly, the 5' and 3' sides of FER were first amplified from the 5' and 3' RACE products of FER, respectively, using the following primer pairs: 5'-AAA ATG GGG TTT GGG AGT GAC-3'/5'-CCA GAA GAT GAA TAC ACT CCA-3' for the 5' side of FER, and 5'-GAC AGC CTG TCT ACA TCA TTA TGG AAC- 3'/5'-CGC CAT CCT GTC ACT ATG TGA G-3' for the 3' side of FER. "
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ABSTRACT: Here, we show that overexpression of fer tyrosine kinase (FER), a non-receptor tyrosine kinase, predicts poor postoperative outcome and might be involved in cancer-cell survival in non-small cell lung cancer (NSCLC). Systematic screening using in silico analyses and quantitative RT-PCR revealed that FER was overexpressed in about 10% of NSCLC patients. Evaluation of FER expression using immunohistochemistry (IHC) on tissue microarrays was consistent with the mRNA level detected using quantitative RT-PCR. In analyses of 135 NSCLC patients who had undergone potential curative resection, we found that FER overexpression detected using IHC had no association with clinicopathological features such as age, sex, smoking history, histological type, disease stage, T factor, N factor, adjuvant chemotherapy history, or EGFR mutation, but was correlated with poor postoperative survival periods. A multivariate Cox regression analysis showed that this prognostic impact was independent of other clinicopathological features. In functional analyses of FER in vitro, FER exhibited a transforming activity, suggesting that it possesses oncogenic functions. We also found that human lung cancer NCI-H661 cells, which exhibited FER-outlier expression, were led to apoptosis by the knockdown of FER using RNA interference. FER overexpression might serve as a prognostic biomarker and be involved in cancer-cell survival in NSCLC.
Available from: onlinelibrary.wiley.com
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ABSTRACT: Previously, we reported that the overexpression of fer tyrosine kinase (FER), a non-receptor tyrosine kinase, is correlated with poor postoperative prognosis and cancer-cell survival in non-small cell lung cancer (NSCLC). In the present study, we further analyzed FER-overexpressed NSCLC cases and identified various patterns of chimeric mRNAs, composed of paraja ring finger 2 (PJA2) and FER. We detected no genomic rearrangements between PJA2 and FER and attributed these chimeric mRNAs to alterations at the transcriptome level: i.e., trans-splicing. Several chimeric patterns were detected concurrently in each patient, and the pattern sets varied among patients, although the pattern in which PJA2 exon 1 was fused to FER exon 3 (designated as Pe1-Fe3 mRNA) was detected constantly. Therefore, in a wide screening for PJA2-FER mRNAs in NSCLC, we focused on this chimeric pattern as a representative chimera. In analyses of 167 NSCLC samples, Pe1-Fe3 mRNA was identified in about 10% of the patients, and the presence of chimeric mRNA was significantly correlated with a high expression level of parental FER mRNA. Furthermore, we found that the detection of Pe1-Fe3 mRNA was correlated with poor postoperative survival periods in NSCLC, consistent with a previous finding in which FER overexpression was correlated with poor postoperative prognosis in NSCLC. This report is the first to suggest a correlation between chimeric mRNA and the expression level of parental mRNA. Furthermore, our findings may be clinically beneficial, suggesting that PJA2-FER mRNAs might serve as a novel prognostic biomarker in NSCLC.
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ABSTRACT: A novel gene (imnp) encoding manganese peroxidase (MnP) from a new and local white-rot fungus, Irpex lacteus F17, was cloned and sequenced. It contains a coding region of 1632 bp, eleven exons and ten introns. The deduced protein, Il-MnP, contains 333 amino acids, with a signal peptide of 26 amino acids. It shows conserved motifs that also exist in other fungal MnPs, including a Mn2+-binding site, Ca2+-binding sites, and eight cysteines. Based on a phylogenetic tree analysis, Il-MnP was deemed to be a new member of the short-type hybrid manganese peroxidases belonging to the class II fungal heme peroxidase subclass/subfamily A.2. Furthermore, the cDNA encoding the mature protein sequence was cloned into the expression vector pET28a and successfully expressed in Escherichia coli Rosetta (DE3). The recombinant protein was refolded and purified to homogeneity, and then partially characterized. Kinetic properties of the recombinant MnP differed from native MnP produced by I. lacteus F17, whereas the spectrum of substrates oxidized by both enzymes was similar. Other attractive features of the recombinant enzyme were its high stability at extreme pH values (from pH 3.5 to 9) and its ability to decolorize a high redox potential azo dye, reactive black 5. The results indicate that this enzyme has a promising biotechnological potential.
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