Analysis of the 3′ UTR of the ART3 and ART4 gene by 3′ inverse RACE-PCR

Abstract

3' Rapid amplification of cDNA ends (3' RACE) is a polymerase chain reaction (PCR) based technique which has been developed to analyse 3' ends of partially known cDNA sequences. To improve the effectiveness of the technique, many investigators have modified the RACE protocol. Here, we describe an alternative procedure for analysing 3' mRNA ends which is based on DNA ligase-mediated self circularization and inverse PCR. This technique is simple and characterized by the exclusive use of gene-specific primers and the absence of unspecific adaptor sequences to obtain highly specific PCR products. We applied the method to analyze the 3' UTR of human mono-ADP-ribosyltransferase (ART) 3 mRNA in testis and heart muscle and of ART4 mRNA in HEL cells. The obtained sequences of ART3 and ART4 mRNA corresponded to data base entries of the respective mRNAs. No adenylate/uridylate-rich elements (AREs) were found in the 3' UTR of ART3 mRNA while one ARE class I motif was detected in the 3' UTR of ART4 mRNA.

The orthologue of the "acatalytic" mammalian ART4 in chicken is an arginine-specific mono-ADP-ribosyltransferase

“…The 3' UTR of the chicken ART4 mRNA was analysed by applying 3' inverse RACE-PCR [ 26 ] or 3' RACE-PCR [ 16 ] on mRNA from HD3 cells using primers derived from the predicted ART4 sequence. As no ART4 specific transcripts were obtained, possibly due to the length of the products we applied conventional RT-PCR analysis to amplify ART4 mRNA reaching into the 3' UTR as far as possible.…”

Stress-induced mobility of OPHIO1 and OPHIO2, DNA transposons of the Dutch elm disease fungi

“…2B), which is the signal recognized by activated CPE-binding proteins (CPEB) thus triggering poly-A elongation (McGrew et al, 1989). Furthermore, a putative UUAUUUUUGCAUUUA motif, only found in OPHIO1, looked strongly like the adenylate-uridylate rich element (ARE) motifs (UUAUUUAAA___AUUUA), an important signal in determining mRNA stability and translation (Friedrich et al, 2005) (underlined with a dotted line in Fig. 2B).…”

“…The Cytoplasmic Poly-A Elongation (CPE) motif (black box) was present in the two 3 0 -UTRs of OPHIO transposons and can trigger poly-A elongation (McGrew et al, 1989). Furthermore, a putative UUAUUUUUGCAUUUA motif (underlined with a dotted line), only found in OPHIO1, demonstrates a high identity with the adenylate-uridylate rich element (ARE) motifs (UUAUUUAAA___AUUUA), involved in mRNA stability and translation (Friedrich et al, 2005). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)…”

Cloning of Full-Length cDNA of Teleost Corticotropin-Releasing Hormone Precursor by Improved Inverse PCR

“…1). Inverse PCR has two advantages: (1) nonspecific PCR products are less likely to be amplified because of the use of two gene-specific primers, 3,5) and (2) both 3 0 -and 5 0 -regions are amplified in a single reaction. In spite of these advantages, inverse PCR has not been as popular as RACE.…”