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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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Mobility shift analysis for DNA binding proteins  recognizing sequences around  δREC. (a) Mobility shift analysis defining a protein complex,  unique to the thymus (arrow),  recognizing the upstream homology region HPS1. (b) Mobility shift gel using the downstream homology region HPS2  as probe. (c) Mobility shift gel  showing reactivity of the identified DNA complex from thymus  to the 48-bp subsegment HPS1A  (arrow). (d) Competitive mobility  shift assay using the 97-bp segment HPS1 as probe. Extracts  used from thymus, spleen, and  HeLa cells were crude and unpurified.
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Figure 2: Mobility shift analysis for DNA binding proteins recognizing sequences around δREC. (a) Mobility shift analysis defining a protein complex, unique to the thymus (arrow), recognizing the upstream homology region HPS1. (b) Mobility shift gel using the downstream homology region HPS2 as probe. (c) Mobility shift gel showing reactivity of the identified DNA complex from thymus to the 48-bp subsegment HPS1A (arrow). (d) Competitive mobility shift assay using the 97-bp segment HPS1 as probe. Extracts used from thymus, spleen, and HeLa cells were crude and unpurified.

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)

Mobility shift analysis for DNA binding proteins  recognizing sequences around  δREC. (a) Mobility shift analysis defining a protein complex,  unique to the thymus (arrow),  recognizing the upstream homology region HPS1. (b) Mobility shift gel using the downstream homology region HPS2  as probe. (c) Mobility shift gel  showing reactivity of the identified DNA complex from thymus  to the 48-bp subsegment HPS1A  (arrow). (d) Competitive mobility  shift assay using the 97-bp segment HPS1 as probe. Extracts  used from thymus, spleen, and  HeLa cells were crude and unpurified.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Mobility shift analysis for DNA binding proteins recognizing sequences around δREC. (a) Mobility shift analysis defining a protein complex, unique to the thymus (arrow), recognizing the upstream homology region HPS1. (b) Mobility shift gel using the downstream homology region HPS2 as probe. (c) Mobility shift gel showing reactivity of the identified DNA complex from thymus to the 48-bp subsegment HPS1A (arrow). (d) Competitive mobility shift assay using the 97-bp segment HPS1 as probe. Extracts used from thymus, spleen, and HeLa cells were crude and unpurified.
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