The two isoforms of mouse terminal deoxynucleotidyl transferase differ in both the ability to add N regions and subcellular localization.
Two alternatively spliced terminal deoxynucleotidyl transferase transcripts, TdTS and TdTL which code respectively for proteins of 509 and 529 amino acids have been previously identified in the mouse thymus. Here we show that the same two transcripts are also present in B lineage cells from bone marrow. In addition we demonstrate that the corresponding 20 amino acid insertion found near the carboxy-terminal end of TdTL significantly alters the function of the enzyme. In contrast to TdTS, TdTL does not catalyse N region insertions at the recombination junction of a V(D)J site-specific recombination substrate. In an attempt to explain the lack of N region insertions we have characterized the different parameters which distinguish the two isoforms of TdT. Examination of transfected cell extracts revealed a reduced capacity of TdTL to add nucleotides to the 3' end of DNA, consistent with a lower terminal transferase activity. Furthermore, the half-life of the TdTL protein in these cells is 2-fold shorter than that of TdTS. Finally, despite the fact that TdTL has the same nuclear localization signal as TdTS, the cellular localization of the two isoforms was strikingly different. In contrast to nuclear TdTS, TdTL was found exclusively in the cytoplasm. All these characteristics could contribute to the functional difference between the two isoforms of TdT. However, the subcellular localization of TdTL on its own can account for its inability to add N regions.
Bentolila
L A
LA
Unité de Génétique et Biochimie du Développement, Paris Cédex 15, France.
Fanton d'Andon
M
M
Nguyen
Q T
QT
Martinez
O
O
Rougeon
F
F
Doyen
N
N
D017398:Alternative Splicing; D000818:Animals; D001402:B-Lymphocytes / Q000201:enzymology; D001483:Base Sequence; D001853:Bone Marrow / Q000201:enzymology*; D002460:Cell Line; D002522:Chlorocebus aethiops; D004253:DNA Nucleotidylexotransferase / Q000096:biosynthesis / Q000378:metabolism*; D017931:DNA Primers; D006412:Hematopoietic Stem Cells / Q000201:enzymology; D007527:Isoenzymes / Q000096:biosynthesis / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology; D013950:Thymus Gland / Q000201:enzymology*; D014162:Transfection
D017931:DNA Primers; D007527:Isoenzymes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004253:DNA Nucleotidylexotransferase