Susan Mendelson

The Nuclear Autoimmune Antigen Ku IS Also Present on the Cell Surface

Polyclonal antibodies were raised against the individual 85 and 70 kDa subunits of the Ku complex purified from nuclear extract prepared from the T cell line MLA144. They specifically recognise the appropriate subunits of the Ku complex from whole cell extract of HeLa cells using Western blot analysis. They are also able to identify the Ku proteins present in the cell membrane using FACS analysis.

Repression of preprotachykinin-A promoter activity is mediated by a proximal promoter element

The rat preprotachykinin-A promoter, which is able to direct reporter gene expression in adult dorsal root ganglia neurons grown in culture, has no detectable activity in HeLa and PC12 cells. DNAase 1 footprinting and electrophoretic mobility shift analyses with HeLa nuclear extract indicated the presence of a protein complex binding to a region of the rat preprotachykinn-A gene promoter between the TATA box and the major transcriptional start site. We demonstrate that the sequence of the preprotachykinin-A promoter spanning nucleotides -47 to +92 functions to repress reporter gene expression in HeLa and PC12 cells but not in adult rat dorsal root ganglia grown in culture, and that this repression is correlated with a protein(s) binding to the element between the TATA box and major transcription initiation site. These results indicate that the tissue-specific expression of the preprotachykinin-A gene could require the interaction of both positive and negative regulatory DNA elements.

Three immediate early gene response elements in the proximal preprotachykinin-A promoter in two functionally distinct domains.

The preprotachykinin-A promoter contains two blocks of DNA sequence, with a high degree of homology to one another, both containing activator protein 1/cAMP response element-like elements which constitute cis-acting regulatory domains. These two domains are differentially regulated in HeLa cells and primary cultures of dorsal root ganglion neurons when they are placed in the context of a reporter gene driven by the c-fos minimum promoter. One of the domains, corresponding to a region of the preprotachykinin promoter spanning nucleotides -345 to -308, contains two activator protein 1 elements adjacent to an E-box binding protein consensus sequence. Both of the activator protein 1 elements can bind a complex containing c-fos/c-fos related antigen proteins and the adjacent E-box element is specifically recognized by proteins present in HeLa nuclear extract. This domain requires the synergistic action of both activator protein 1 elements to drive expression of the reporter gene in both HeLa and dorsal root ganglion cells. The second or proximal domain spans nucleotides -198 to -155 and contains a previously characterized activator protein 1/cAMP response element/ATF enhancer element which, in contrast to the activator protein 1 elements in the distal domain, functions in both HeLa and dorsal root ganglion cells as one copy. This domain is differentially regulated in HeLa and dorsal root ganglia. The previously characterized enhancer activity is repressed in the context of the extended cis-acting domain in HeLa cells but remains active in dorsal root ganglion, although no further enhancement of activity supported by the single enhancer is observed when in the context of the extended sequence. This proximal domain, in addition to binding the enhancer complex, can be bound by at least two other complexes, one of which binds to an E-box consensus sequence. As the elements corresponding to the E-box consensus in both domains cross-compete for binding of specific complex(es) it would appear that repression of the activity of the proximal domain is correlated with a specific protein complex binding adjacent to the characterized enhancer in the region spanning nucleotides -198 to -155. The preprotachykinin-A proximal promoter is therefore bound by multiple activator protein I complexes, which in the context of the cis-acting domains in which they are present can be differentially regulated. In the proximal domain their function may also be regulated in a tissue-specific manner by other proteins which bind to adjacent regulatory elements.