Abstract
A novel Ets protein was isolated by yeast one-hybrid screening of a cDNA library made from lipopolysaccharide-stimulated mouse splenic B cells, using the SP6 kappa promoter kappaY element as a bait. The novel Ets protein was most closely related to PU.1 and Spi-B within the DNA binding Ets domain and was therefore named Spi-C. However, Spi-C may represent a novel subgroup within the Ets protein family, as it differed significantly from Spi-B and PU.1 within helix 1 of the Ets domain. Spi-C was encoded by a single-copy gene that was mapped to chromosome 10, region C. Spi-C interacted with DNA similarly to PU.1 as judged by methylation interference, band-shift and site selection analysis, and activated transcription of a kappaY element reporter gene upon co-transfection of HeLa cells. Spi-C RNA was expressed in mature B lymphocytes and at lower levels in macrophages. Furthermore, pre-B cell and plasma cell lines were Spi-C-negative, suggesting that Spi-C might be a regulatory molecule during a specific phase of B lymphoid development.
Highlights
Proteins that are members of the Ets family of transcription factors are involved in a variety of developmental and cellular responses [1]
The protein is mainly expressed in the peripheral B lymphoid compartment; it is not expressed in early B cell precursors or in pre-B cell lines and appears to be down-regulated as the B cell matures to the plasma cell stage
In an effort to identify the proteins that interact with these DNA elements, we used the sequence shown in Fig. 1A as bait in a yeast one-hybrid screen of a cDNA library made from LPS-stimulated mouse B cells (Fig. 1B)
Summary
Yeast Strains—Four copies of a fragment containing the pentadecamer element and the Y core element [16, 24] were cloned into the pHisi and pLacZi vectors (CLONTECH), between the XbaI and EcoRI sites, and SmaI and EcoRI sites, respectively. The integration was verified by Southern blotting of PCR1-amplified DNA from genomic yeast DNA using either Hisi- or LacZi-specific primers, using one copy of the inserted fragment as a probe (5Ј-TACTCTCAAACAGCTGTGTAATTTACTTTCC-3Ј). CDNA Library Screening and Sequencing—The Hybri-ZAP library was spread on 90-mm plates at 22,000 plaque-forming units/plate and overlaid with sodium chloride magnesium to obtain 60 phage pools These were screened via PCR using two Spi-C-specific primers (5Ј-GCAAACATTTCAAGACGCC-3Ј and 5Ј-CTGTACGGATTGGTGGAAGC3Ј), the products separated by electrophoresis and blotted onto charged nylon membranes, which were probed with a polynucleotide kinase end-labeled oligonucleotide (5Ј-CAGACCTGTATTTGGAAGGA-3Ј) as described by the manufacturer (Bio-Rad). Two positive pools were identified, and these were subsequently screened according to standard procedures using the original yeast Spi-C clone as probe and excised as plasmids according to the manufacturer’s instructions (Stratagene). FISH signals and DAPI banding pattern were recorded separately by photography, and the assignment of the FISH mapping data with chromosomal bands was achieved by superimposing FISH signals with DAPI-banded chromosomes [30]
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