Cloning of R kappa B, a novel DNA-binding protein that recognizes the interleukin-2 receptor alpha chain kappa B site.

Transcriptional activation of the interleukin-2 receptor alpha (IL-2R alpha) gene in T cells is dependent on a regulatory element that can bind NF-kappa B but differs in sequence and function from the kappa B site of the immunoglobulin (Ig) enhancer. To define the molecular basis of gene-specific regulation by this variant kappa B site, we have used electrophoretic mobility shift assays to characterize a novel gene product, designated R kappa B, that binds preferentially to the related kappa B site in IL-2R alpha. A cDNA encoding this 107-kD protein has been isolated from a lambda gt11 expression library by screening with a probe containing the IL-2R alpha kappa B site. R kappa B is a tissue-specific transcription factor that contains an amino acid sequence similar to a discrete region of the myogenic regulatory protein MyoD, but is unrelated to other DNA-binding proteins, including those belonging to the rel/dorsal gene family. This novel kappa B binding protein may therefore contribute to the regulation of distinct cellular and viral genes with variant kappa B sites.

Lymphoid expression and TATAA binding of a human protein containing an Antennapedia homeodomain.

In an effort to identify human proteins that bind to the TATAA box, a lambda gt-11 expression library was screened with a radiolabeled DNA probe containing 12 copies of the TATAA sequence. A cDNA encoding a specific TATAA binding protein was isolated and found to contain a homeobox domain identical at 59 of 60 residues to the Drosophila Antennapedia (Antp) homeodomain, as well as another conserved motif found in homeotic genes, the homeo-specific pentapeptide. Although this and other Antp-like homeobox proteins have been described previously in neuronal cells and fibroblasts, we report the expression of this gene in lymphoid cells. This cDNA, isolated from a B-cell library, hybridizes to a 1.6-kb messenger RNA in several T- and B-cell lines, and the expected protein was identified in Jurkat T-lymphoid cells by Western blot analysis. The DNA binding specificity of this human Antp clone was analyzed using single-base mutations of the TATAA sequence. The first thymidine, as well as the last three bases (TAA), were important for homeobox binding. Finally, the function of the highly conserved homeospecific pentapeptide protein region was investigated in both the human and Drosophila Antp proteins. The homeospecific pentapeptide region was not required for DNA binding, and Drosophila Antp proteins mutated in the pentapeptide region were able to transactivate the Ubx promoter in Schneider L2 cells, in contrast to a homeodomain mutation, suggesting an alternative function for the homeospecific pentapeptide in homeotic genes. Because the human Antp TATAA binding protein is expressed in both lymphoid and non-lymphoid cells, we suggest that this homeobox gene has evolved a more general transcriptional regulatory function in higher eukaryotic cells.

Isolation of a cDNA clone encoding a zinc finger protein highly expressed in T-leukemia lines.

A cDNA clone encoding a novel zinc finger protein expressed in lymphoid cells has been isolated. This protein contains 5 repeats of the C2H2 motif previously described in the Drosophila gap gene, Krüppel, which is involved in embryo segmentation. Northern blot analysis showed that the messenger RNA (mRNA) encoding this protein is expressed at high levels in a variety of T-leukemia cell lines, at lower levels in some B cells, but is not observed in nonlymphoid cells. Within the T lineage, the mRNA is found at high levels in both alpha beta and gamma delta T cells. These data suggest that this cDNA, designated Hkr-T1, represents a gene that may contribute to the determination of the differentiation and the specificity within lymphoid cells.