Alternative forms of the human thioredoxin mRNA: identification and characterization.
Thioredoxin (TRX) is an ubiquitous and relatively conserved oxidoreductant enzyme which is involved in a multitude of redox reactions through the formation of reversible disulfide bonds. A recent report indicates the presence of novel isoforms of TRX proteins isolated from MP6 cell lines [Rosén et al., Int. Immunol. 7 (1995) 625-633]. In these isoforms, as evidenced from amino acid sequencing, several Lys residues of the wild-type sequence were replaced by Arg. Although the human genome contains several (isoformic) copies of the TRX gene, only one appears to be transcriptionally active [Kaghad et al., Gene, 140 (1994) 273-278]. As we characterized the isoforms of TRX mRNAs, we found that several MP6 TRX cDNA clones were devoid of the characteristic poly(A) tail. In order to increase the efficiency of isolating mRNAs without the poly(A) tail, we developed a novel procedure for exclusive capturing of a specific mRNA by magnetic beads coated with biotinylated antisense oligodeoxyribonucleotide. Using this method on MP6 cell total RNA, we isolated an additional truncated version of the TRX mRNA. This latter form does not produce any variant TRX enzyme, as an inframe stop codon truncates the product. This isoform was also present in mRNAs isolated from human placenta, leucocyte cells and Molt4 cells, the latter two being the progenitors of MP6 cells. From a thorough analysis of the sequence of the truncated TRX mRNA, we conclude that this variant originated from an event of altered splicing, as consensus splice sites were present in the normal TRX cDNA at precise positions.
Hariharan
J
J
Astra Research Centre India, Malleswaram, Bangalore, India.
Hebbar
P
P
Ranie
J
J
Philomena
Sinha
A M
AM
Datta
S
S
D017398:Alternative Splicing; D001483:Base Sequence; D018076:DNA, Complementary; D015723:Gene Library; D006801:Humans; D008280:Magnetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification; D013879:Thioredoxins / Q000235:genetics*
D018076:DNA, Complementary; D012333:RNA, Messenger; D013879:Thioredoxins