Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In vitro studies have demonstrated that deoxycytidine kinase (dCK) plays a crucial role in the mechanism of resistance to cytarabine (AraC). The resistant phenotype in vitro is always a result of mutational inactivation of dCK, leading to defects in the metabolic pathways of AraC. Although inactivation of dCK has shown to be one of the major mechanism of resistance to AraC in vitro, limited in vivo data are available. To improve research concerning the involvement of dCK inactivation in patients with acute myeloid leukemia (AML), we have set up a protocol that allows direct assessment of dCK expression and activity in primary human cells. In this protein activity truncation assay (PAT assay), the complete coding region of dCK is amplified by RT-PCR and a T7 RNA polymerase promoter sequence is introduced upstream of the coding region in a nested PCR reaction. After in vitro transcription-translation dCK proteins are analyzed for their molecular weight and phosphorylating capacities. We show that this relatively quick method can be used in purified, primary human leukemic blasts. In addition, inactivation of dCK by point mutations, deletions or genomic rearrangements can easily be detected in AraC-resistant cell lines. This novel assay may contribute to further elucidate the mechanism of AraC resistance in vivo.
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PMID:A novel RT-PCR-based protein activity truncation assay for direct assessment of deoxycytidine kinase in small numbers of purified leukemic cells. 1136 49

Alternative polyadenylation has been demonstrated as a tier of gene expression regulation in eukaryotes. However, its role has not been elucidated at the cellular level. Equipped with techniques to isolate single cells by fluorescence-activated cell sorting (FACS) and laser captured micro-dissection, analysis of alternative polyadenylation in specific cell types becomes possible. We present a method to generate poly(A) tags for high-throughput sequencing (PAT-seq) libraries from very low amount of total RNA. This protocol targets the junction of the 3'-UTR and poly(A) tail of transcripts. Ten nanograms of total RNA isolated from the FACS-sorted cells was reverse-transcribed to double stranded cDNA with a anchored oligo dT(18) primer containing maximal T7 promoter sequence. Then, an RNA amplification step using in vitro transcription of T7 RNA polymerase was carried out. Achieved cRNA was fragmented by partial digestion. First strand synthesis was carried out by using a partial adaptor sequence with random 9-nt primer to introduce the adaptor at the 5' end. An anchored oligo dT primer containing adaptor sequence on 3' end was introduced through second strand cDNA synthesis. This new method has been applied to investigate polyadenylation using nanogram amount of total RNA from Arabidopsis cells.
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PMID:Poly(A) tag library construction from 10 ng total RNA. 2548 14