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Identification of a DNA segment exhibiting rearrangement modifying effects upon transgenic delta-deleting elements.

Janowski KM, Ledbetter S, Mayo MS, Hockett RD - J. Exp. Med. (1997)

Bottom Line: Furthermore, HPS1A augments recombination frequency of extrachromosomal substrates in an in vitro recombination assay.DNA binding proteins recognizing HPS1A have been identified and are restricted to early B and T cells, during the time of active rearrangement of endogenous TCR and immunoglobulin loci.These data are consistent with delta deletion playing an important role in maintaining separate TCR alpha and delta loci.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama 35233-7331, USA.

ABSTRACT
Control of the rearrangement and expression of the T cell receptor alpha and delta chains is critical for determining T cell type. The process of delta deletion is a candidate mechanism for maintaining separation of the alpha and delta loci. Mice harboring a transgenic reporter delta deletion construct show alpha/beta T cell lineage-specific use of the transgenic elements. A 48-basepair segment of DNA, termed HPS1A, when deleted from this reporter construct, loses tight lineage-specific rearrangement control of transgenic elements, with abundant rearrangements of transgenic delta-deleting elements now in gamma/delta T cells. Furthermore, HPS1A augments recombination frequency of extrachromosomal substrates in an in vitro recombination assay. DNA binding proteins recognizing HPS1A have been identified and are restricted to early B and T cells, during the time of active rearrangement of endogenous TCR and immunoglobulin loci. These data are consistent with delta deletion playing an important role in maintaining separate TCR alpha and delta loci.

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Sequence of human  and murine δREC. (a) Comparison of human δREC and murine  δREC1. Human sequence is  shown on the top line, murine  sequence on the bottom. Vertical lines represent identity between the two sequences; dashes  represent gaps in the sequence  placed by the analysis software  for better fit. The canonical h-s-n  recombinase recognition motif  is boxed and labeled. The dark  underlines outline the two areas  of ∼80% identity between human and mouse, HPS1 and  HPS2, respectively. (b) HPS1 has  been broken into the two areas  HPS1A and HPS1B. Human sequence is on the top, murine sequence on the bottom. The bracket outlines the area of HPS1A that is protected during DNAse I footprinting  (footprint not shown). These sequence data are available from EMBL/ GenBank/DDBJ under accession numbers Y13607Y13607 and Y13608Y13608.
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Figure 1: Sequence of human and murine δREC. (a) Comparison of human δREC and murine δREC1. Human sequence is shown on the top line, murine sequence on the bottom. Vertical lines represent identity between the two sequences; dashes represent gaps in the sequence placed by the analysis software for better fit. The canonical h-s-n recombinase recognition motif is boxed and labeled. The dark underlines outline the two areas of ∼80% identity between human and mouse, HPS1 and HPS2, respectively. (b) HPS1 has been broken into the two areas HPS1A and HPS1B. Human sequence is on the top, murine sequence on the bottom. The bracket outlines the area of HPS1A that is protected during DNAse I footprinting (footprint not shown). These sequence data are available from EMBL/ GenBank/DDBJ under accession numbers Y13607Y13607 and Y13608Y13608.

Mentions: A candidate region for controlling lineage specificity in TG1 was discovered by first comparing sequences between human and murine δREC. The rationale was to search for areas of high DNA homology between δRECs, and then look for DNA binding proteins in the developing T cells of the thymus. Two areas of very high sequence homology, outside of the h-s-n canonical recombination motif, were noted 5′ and 3′ to the recombination signal (Fig. 1 a; 20). Nuclear extracts from murine thymus were used as probes to determine reactivity by mobility shift assay to both regions. DNA binding proteins were found in murine thymus to a 97-bp upstream region, designated HPS1, that is not detected in mature T cells of the spleen, nor in a HeLa cell nuclear extract (Fig. 2 a, arrow).


Identification of a DNA segment exhibiting rearrangement modifying effects upon transgenic delta-deleting elements.

Janowski KM, Ledbetter S, Mayo MS, Hockett RD - J. Exp. Med. (1997)

Sequence of human  and murine δREC. (a) Comparison of human δREC and murine  δREC1. Human sequence is  shown on the top line, murine  sequence on the bottom. Vertical lines represent identity between the two sequences; dashes  represent gaps in the sequence  placed by the analysis software  for better fit. The canonical h-s-n  recombinase recognition motif  is boxed and labeled. The dark  underlines outline the two areas  of ∼80% identity between human and mouse, HPS1 and  HPS2, respectively. (b) HPS1 has  been broken into the two areas  HPS1A and HPS1B. Human sequence is on the top, murine sequence on the bottom. The bracket outlines the area of HPS1A that is protected during DNAse I footprinting  (footprint not shown). These sequence data are available from EMBL/ GenBank/DDBJ under accession numbers Y13607Y13607 and Y13608Y13608.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198968&req=5

Figure 1: Sequence of human and murine δREC. (a) Comparison of human δREC and murine δREC1. Human sequence is shown on the top line, murine sequence on the bottom. Vertical lines represent identity between the two sequences; dashes represent gaps in the sequence placed by the analysis software for better fit. The canonical h-s-n recombinase recognition motif is boxed and labeled. The dark underlines outline the two areas of ∼80% identity between human and mouse, HPS1 and HPS2, respectively. (b) HPS1 has been broken into the two areas HPS1A and HPS1B. Human sequence is on the top, murine sequence on the bottom. The bracket outlines the area of HPS1A that is protected during DNAse I footprinting (footprint not shown). These sequence data are available from EMBL/ GenBank/DDBJ under accession numbers Y13607Y13607 and Y13608Y13608.
Mentions: A candidate region for controlling lineage specificity in TG1 was discovered by first comparing sequences between human and murine δREC. The rationale was to search for areas of high DNA homology between δRECs, and then look for DNA binding proteins in the developing T cells of the thymus. Two areas of very high sequence homology, outside of the h-s-n canonical recombination motif, were noted 5′ and 3′ to the recombination signal (Fig. 1 a; 20). Nuclear extracts from murine thymus were used as probes to determine reactivity by mobility shift assay to both regions. DNA binding proteins were found in murine thymus to a 97-bp upstream region, designated HPS1, that is not detected in mature T cells of the spleen, nor in a HeLa cell nuclear extract (Fig. 2 a, arrow).

Bottom Line: Furthermore, HPS1A augments recombination frequency of extrachromosomal substrates in an in vitro recombination assay.DNA binding proteins recognizing HPS1A have been identified and are restricted to early B and T cells, during the time of active rearrangement of endogenous TCR and immunoglobulin loci.These data are consistent with delta deletion playing an important role in maintaining separate TCR alpha and delta loci.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama 35233-7331, USA.

ABSTRACT
Control of the rearrangement and expression of the T cell receptor alpha and delta chains is critical for determining T cell type. The process of delta deletion is a candidate mechanism for maintaining separation of the alpha and delta loci. Mice harboring a transgenic reporter delta deletion construct show alpha/beta T cell lineage-specific use of the transgenic elements. A 48-basepair segment of DNA, termed HPS1A, when deleted from this reporter construct, loses tight lineage-specific rearrangement control of transgenic elements, with abundant rearrangements of transgenic delta-deleting elements now in gamma/delta T cells. Furthermore, HPS1A augments recombination frequency of extrachromosomal substrates in an in vitro recombination assay. DNA binding proteins recognizing HPS1A have been identified and are restricted to early B and T cells, during the time of active rearrangement of endogenous TCR and immunoglobulin loci. These data are consistent with delta deletion playing an important role in maintaining separate TCR alpha and delta loci.

Show MeSH
Related in: MedlinePlus