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The biochemical activities that underlie the genetically defined activator and repressor functions of the VIVIPAROUSl (VP1) protein have resisted in vitro analysis. Here, we show that a glutathione S-transferase (GST) fusion protein, including only the highly conserved 8 3 domain of VP1, has a highly cooperative, sequence-specific DNA binding activity. GST fusion proteins that include larger regions of the VP1 protein have very low activity, indicating that removal of the flanking protein sequences is necessary to elicit DNA binding in vitro. DNA competition and DNase I footprinting analyses show that B3 binds specifically to the Sph element involved in VP1 activation of the Cf gene, whereas binding to the G-box-type VP1-responsive element is of low affinity and is nonspecific. Footprint analysis of the Cf promoter revealed that sequences flanking the core TCCATGCAT motif of Sph also contribute to the recognition of the Sph element in its native context. The salient features of the in vitro GST-B3 DNA interaction are in good agreement with the protein and DNA sequence requirements defined by the functional analyses of VP1 and VP1-responsive elements in maize cells.

A culture method to grow two morphologically distinguishable normal human breast epithelial cell types derived from reduction mammoplasty has been developed. Type I cells were characterized by a more variable cell shape, smooth cell colony boundaries, the expression of epithelial membrane antigen (EMA) and keratin 18 and the non-expression of keratin 14 and alpha 6 integrin. In addition, the Type I cells were growth stimulated by fetal bovine serum (FBS) and were deficient in gap junctional intercellular communication (GJIC). In contrast, Type II cells were characterized by a uniform cell shape, expression of keratin 14 and alpha 6 integrin and the non-expression of EMA and keratin 18. In addition, Type II cells were growth inhibited by FBS and were proficient in GJIC. Type I cells can be induced by cholera toxin to change their morphology to a Type II cell morphology. Hence, Type I cells antigenically resemble luminal epithelial cells, while the Type II cells more closely resemble basal epithelial cells. Type I and Type II cells were transfected with SV40 DNA. Clones with extended lifespans were obtained from both Type I and Type II cells by SV40 transfection. Some (2/9) of the SV40-transfected Type I cell clones became immortal (> 100 cumulative population doubling level), whereas none (0/8) of the SV40-transfected Type II cell clones became immortal. The SV-40-transfected Type I and Type II cell-derived extended life clones and immortal cell lines phenotypically resembled their parental cells with respect to EMA, keratin 14 and keratin 18 expression and GJIC. Each (9/9) of the SV40 transfected Type I cell clones grew in soft agar; none (0/8) of the SV40-transfected Type II cell clones were capable of growing in soft agar. These results provide evidence that normal human breast epithelial cells, derived from reduction mammoplasty, can be separated into two morphologically and antigenically different cell types and that these two different cell types significantly differ in their response to an oncogenic (SV40) stimulus.

Transcription factor IID (TFIID) binds to the TATA box promoter element and regulates the expression of most eukaryotic genes transcribed by RNA polymerase II. Complementary DNA (cDNA) encoding a human TFIID protein has been cloned. The human TFIID polypeptide has 339 amino acids and a molecular size of 37,745 daltons. The carboxyl-terminal 181 amino acids of the human TFIID protein shares 80% identity with the TFIID protein from Saccharomyces cerevisiae. The amino terminus contains an unusual repeat of 38 consecutive glutamine residues and an X-Thr-Pro repeat. Expression of DNA in reticulocyte lysates or in Escherichia coli yielded a protein that was competent for both DNA binding and transcription activation.

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