B-cell factor 1 is required for optimal expression of the DRA promoter in B cells.

The X box in the DRA promoter of the human histocompatibility complex is required for expression of the DRA gene in B cells. We show that a B-cell factor binds to a sequence that is clearly distinguishable from binding sites for the previously described X box binding nuclear proteins RF-X, NF-X, NF-Xc, NF-S, hXBP, and AP-1. Mutations in the DRA X box that disrupt the binding of this factor result in a lower level of gene expression, as does the presence of Id (a trans-dominant regulatory protein that negatively regulates helix-loop-helix proteins). Furthermore, this factor is recognized by antibodies directed against the helix-loop-helix protein A1, a mouse homolog of the immunoglobulin enhancer binding proteins E12/E47, and it binds to sequences in other genes that were previously shown to bind these proteins. By these criteria, this factor is BCF-1.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

A technique for the short term organ culture of larval salivary glands of D. melanogaster is described. Cultured Puff Stage 1 glands respond to 20-OH ecdysone by initiating the cycle of puffing activity characteristic of late larval development and puparium formation. This puffing cycle involves the sequential activation of at least 125 puffs. Their response to ecdysone allows these puffs to be divided into 3 main classes: a) PS1 puffs that regress (e.g. 25AC); b) puffs activated very rapidly (within 5 min) (e.g. 23E, 74EF, 75B) and c) puffs activated only after longer periods (>4 h) (e.g. 62E, 78D, 22C, 63E and 82F). The detailed behaviour of representatives of each class is described. These data support Clever's distinction of ‘early’ and ‘late’ ecdysone responsive sites.     

Expression of a 91-kilodalton PEA3-binding protein is down-regulated during differentiation of F9 embryonal carcinoma cells.

Proteins binding to the PEA3 enhancer motif (AGGAAG) activate the polyomavirus early promoter and help comprise the viral late mRNA initiator element (W. Yoo, M. E. Martin, and W. R. Folk, J. Virol. 65:5391-5400, 1991). Because many developmentally regulated cellular genes have PEA3 motifs near their promoter sequences, and because Ets family gene products activate the PEA3 motif, we have studied the expression of PEA3-binding proteins and Ets-related proteins during differentiation of F9 embryonal carcinoma cells. An approximately 91-kDa protein (PEA3-91) was identified in F9 cell nuclear extracts by UV cross-linking to a radiolabeled PEA3 oligonucleotide probe, and expression of PEA3-91 was down-regulated after differentiation of F9 cells to parietal endoderm. The c-ets-1 gene product binds to a sequence in the murine sarcoma virus long terminal repeat that is similar to the PEA3 motif (cGGAAG), but PEA3-91 was not cross-linked to this Ets-1-binding motif, nor did antiserum which recognizes murine c-ets-1 and c-ets-2 proteins have any effect on PEA3-binding activity in mobility shift assays. Furthermore, c-ets-1 mRNA was not detected in undifferentiated or differentiated F9 cells, and c-ets-2 mRNA levels remained high after differentiation. Antiserum against the Drosophila Ets-related E74A protein, however, recognized an approximately 92-kDa protein in F9 cells whose expression during differentiation varied in a manner identical to that of PEA3-91. These data suggest that PEA3-91 is not the product of the ets-1 or ets-2 genes but is likely to be the product of a murine homolog of the Drosophila E74 gene.     

The sequence specificity of homeodomain-DNA interaction

The Drosophila developmental gene, engrailed, encodes a sequence-specific DNA binding activity. Using deletion constructs expressed as fusion proteins in E. coli, we localized this activity to the conserved homeodomain (HD). The binding site consensus, TCAATTAAAT, is found in clusters in the engrailed regulatory region. Weak binding of the En HD to one copy of a synthetic consensus is enhanced by adjacent copies. The distantly related HD encoded by fushi tarazu binds to the same sites as the En HD, but differs in its preference for related sites. Both HDs bind a second type of sequence, a repeat of TAA. The similarity in sequence specificity of En and Ftz HDs suggests that, within families of DNA binding proteins, close relatives will exhibit similar specificities. Competition among related regulatory proteins might govern which protein occupies a given binding site and consequently determine the ultimate effect of cis-acting regulatory sites.     

Nuclear factor EF-1A binds to the adenovirus E1A core enhancer element and to other transcriptional control regions.

We have identified a cellular enhancer-binding protein, present in nuclear extracts prepared from human and rodent cells, that binds to the adenovirus E1A enhancer element I sequence. The factor has been termed EF-1A, for enhancer-binding factor to the E1A core motif. EF-1A was found to bind to two adjacent, related sequence motifs in the E1A enhancer region (termed sites A and B). The binding of EF-1A to these adjacent sites, or to synthetic dimerized sites of either motif, was cooperative. The cooperative binding of EF-1A to these sites was not subject to strict spacing constraints. EF-1A also bound to related sequences upstream of the E1A enhancer region and in the polyomavirus and adenovirus E4 enhancer regions. The EF-1A-binding region in the E1A enhancer stimulated expression of a linked gene in human 293 cells when multimerized. Based on the contact sites for EF-1A binding determined by chemical interference assays, this protein appears to be distinct from any previously characterized nuclear binding protein.