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The long isoform of terminal deoxynucleotidyl transferase enters the nucleus and, rather than catalyzing nontemplated nucleotide addition, modulates the catalytic activity of the short isoform.

Benedict CL, Gilfillan S, Kearney JF - J. Exp. Med. (2001)

Bottom Line: Two splice variants of Tdt have been described, but only one (short isoform of Tdt [TdtS]) has been convincingly demonstrated to catalyze nontemplated (N) addition in vitro.In contrast to previous in vitro results, both TdtS and TdtL are translocated to the nucleus in our model.We conclude that TdtL localizes to the nucleus in vivo where it serves to modulate TdtS function.

View Article: PubMed Central - PubMed

Affiliation: Division of Developmental and Clinical Immunology, Department of Microbiology, University of Alabama at Birmingham, Alabama 35294, USA.

ABSTRACT
During variable/diversity/joining (V[D]J) recombination, the enzyme terminal deoxynucleotidyl transferase (Tdt) adds random nucleotides at the junctions of the rearranging gene segments, increasing diversity of the antibody (Ab) and T cell receptor repertoires. Two splice variants of Tdt have been described, but only one (short isoform of Tdt [TdtS]) has been convincingly demonstrated to catalyze nontemplated (N) addition in vitro. We have expressed each splice variant of Tdt in transgenic (Tg) mice and found that the TdtS transgene catalyzes N addition on the endogenous Tdt(-/)- background and in fetal liver, but that the long isoform of Tdt (TdtL) transgene does neither. In contrast to previous in vitro results, both TdtS and TdtL are translocated to the nucleus in our model. Furthermore, TdtL/TdtS double Tg mice exhibit less N addition in fetal liver than do TdtS Tg mice. Whereas the TdtS transgene was shown to have functional consequences on the antiphosphorylcholine (PC) B cell repertoire, TdtL Tg mice exhibit a normal PC response, and Tdt(-/)- mice actually exhibit an increase in the PC response and in TEPC 15 idiotype(+) Ab production. We conclude that TdtL localizes to the nucleus in vivo where it serves to modulate TdtS function.

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Immunofluorescence of Tdt Tg splenocytes by fluorescence microscopy. Immunofluorescence of LPS-activated adult spleen B cells from (A) non-Tg LM, (B) line V (TdtS), (C) line I, (D) line B, and (E) line H (TdtL) Tg mice, stained for cytoplasmic μ chains (red) and Tdt (green). All Tg lines exhibit nuclear staining for Tdt. Original magnification indicated by bar inset.
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Figure 2: Immunofluorescence of Tdt Tg splenocytes by fluorescence microscopy. Immunofluorescence of LPS-activated adult spleen B cells from (A) non-Tg LM, (B) line V (TdtS), (C) line I, (D) line B, and (E) line H (TdtL) Tg mice, stained for cytoplasmic μ chains (red) and Tdt (green). All Tg lines exhibit nuclear staining for Tdt. Original magnification indicated by bar inset.

Mentions: Because there was no prior evidence for TdtL protein expression in vivo, we screened our TdtL Tg mouse lines by immunofluorescent staining of LPS-stimulated splenocytes, the method that previously yielded the greatest abundance and most easily detected Tg protein in our TdtS Tg lines. Using this approach, TdtL protein was detected in 7 of our 10 independent TdtL Tg lines, three of which (lines I, B, and H) were chosen for detailed study. By immunofluorescence microscopy, TdtL Tg B cells appeared to express TdtL in the nucleus similarly to the nuclear staining seen in TdtS Tg B cells (line V; Fig. 2). These data are in direct contrast to in vitro expression data of Bentolila et al. 25 who demonstrated by confocal microscopy that TdtL was excluded from the nucleus. In our experiments, the TdtL Tg splenocytes appear to stain more brightly for cytoplasmic Tdt expression than do TdtS cells, but TdtL is not excluded from the nucleus in any of our TdtL Tg lines. Non-Tg LM control splenocytes were negative for Tdt expression in both the nucleus and the cytoplasm after LPS stimulation (Fig. 2). Additionally, we screened for TdtL expression in newborn liver (Fig. 1 B), a tissue in which our TdtS transgene was expressed whereas endogenous Tdt is not (27). We found that TdtL was also expressed in the nucleus of fetal liver cells of TdtL lines B, I, and H, although there were apparent differences in the levels of expression. By measuring the mean fluorescence intensity of TdtL staining by density slicing, we found that H and I expressed two- to threefold higher levels of Tdt than did line B (Fig. 3). These findings opened the possibility that, if functional, TdtL might affect the development of the adult B cell repertoire as did expression of our TdtS transgene.


The long isoform of terminal deoxynucleotidyl transferase enters the nucleus and, rather than catalyzing nontemplated nucleotide addition, modulates the catalytic activity of the short isoform.

Benedict CL, Gilfillan S, Kearney JF - J. Exp. Med. (2001)

Immunofluorescence of Tdt Tg splenocytes by fluorescence microscopy. Immunofluorescence of LPS-activated adult spleen B cells from (A) non-Tg LM, (B) line V (TdtS), (C) line I, (D) line B, and (E) line H (TdtL) Tg mice, stained for cytoplasmic μ chains (red) and Tdt (green). All Tg lines exhibit nuclear staining for Tdt. Original magnification indicated by bar inset.
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Related In: Results  -  Collection

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Figure 2: Immunofluorescence of Tdt Tg splenocytes by fluorescence microscopy. Immunofluorescence of LPS-activated adult spleen B cells from (A) non-Tg LM, (B) line V (TdtS), (C) line I, (D) line B, and (E) line H (TdtL) Tg mice, stained for cytoplasmic μ chains (red) and Tdt (green). All Tg lines exhibit nuclear staining for Tdt. Original magnification indicated by bar inset.
Mentions: Because there was no prior evidence for TdtL protein expression in vivo, we screened our TdtL Tg mouse lines by immunofluorescent staining of LPS-stimulated splenocytes, the method that previously yielded the greatest abundance and most easily detected Tg protein in our TdtS Tg lines. Using this approach, TdtL protein was detected in 7 of our 10 independent TdtL Tg lines, three of which (lines I, B, and H) were chosen for detailed study. By immunofluorescence microscopy, TdtL Tg B cells appeared to express TdtL in the nucleus similarly to the nuclear staining seen in TdtS Tg B cells (line V; Fig. 2). These data are in direct contrast to in vitro expression data of Bentolila et al. 25 who demonstrated by confocal microscopy that TdtL was excluded from the nucleus. In our experiments, the TdtL Tg splenocytes appear to stain more brightly for cytoplasmic Tdt expression than do TdtS cells, but TdtL is not excluded from the nucleus in any of our TdtL Tg lines. Non-Tg LM control splenocytes were negative for Tdt expression in both the nucleus and the cytoplasm after LPS stimulation (Fig. 2). Additionally, we screened for TdtL expression in newborn liver (Fig. 1 B), a tissue in which our TdtS transgene was expressed whereas endogenous Tdt is not (27). We found that TdtL was also expressed in the nucleus of fetal liver cells of TdtL lines B, I, and H, although there were apparent differences in the levels of expression. By measuring the mean fluorescence intensity of TdtL staining by density slicing, we found that H and I expressed two- to threefold higher levels of Tdt than did line B (Fig. 3). These findings opened the possibility that, if functional, TdtL might affect the development of the adult B cell repertoire as did expression of our TdtS transgene.

Bottom Line: Two splice variants of Tdt have been described, but only one (short isoform of Tdt [TdtS]) has been convincingly demonstrated to catalyze nontemplated (N) addition in vitro.In contrast to previous in vitro results, both TdtS and TdtL are translocated to the nucleus in our model.We conclude that TdtL localizes to the nucleus in vivo where it serves to modulate TdtS function.

View Article: PubMed Central - PubMed

Affiliation: Division of Developmental and Clinical Immunology, Department of Microbiology, University of Alabama at Birmingham, Alabama 35294, USA.

ABSTRACT
During variable/diversity/joining (V[D]J) recombination, the enzyme terminal deoxynucleotidyl transferase (Tdt) adds random nucleotides at the junctions of the rearranging gene segments, increasing diversity of the antibody (Ab) and T cell receptor repertoires. Two splice variants of Tdt have been described, but only one (short isoform of Tdt [TdtS]) has been convincingly demonstrated to catalyze nontemplated (N) addition in vitro. We have expressed each splice variant of Tdt in transgenic (Tg) mice and found that the TdtS transgene catalyzes N addition on the endogenous Tdt(-/)- background and in fetal liver, but that the long isoform of Tdt (TdtL) transgene does neither. In contrast to previous in vitro results, both TdtS and TdtL are translocated to the nucleus in our model. Furthermore, TdtL/TdtS double Tg mice exhibit less N addition in fetal liver than do TdtS Tg mice. Whereas the TdtS transgene was shown to have functional consequences on the antiphosphorylcholine (PC) B cell repertoire, TdtL Tg mice exhibit a normal PC response, and Tdt(-/)- mice actually exhibit an increase in the PC response and in TEPC 15 idiotype(+) Ab production. We conclude that TdtL localizes to the nucleus in vivo where it serves to modulate TdtS function.

Show MeSH
Related in: MedlinePlus