Iris A. Mastrangelo

Synergistic transcriptional activation of the MCK promoter by p53: tetramers link separated DNA response elements by DNA looping

The WAF1, Cyclin G and muscle creatine kinase (MCK) genes, all contain multiple copies of the consensus p53-binding element within their regulatory regions. We examined the role of these elements in transactivation of the muscle creatine kinase (MCK) gene by p53. The MCK promoter possesses distal (−3182 to −3133) and proximal (−177 to −81) p53-binding elements within which residues −3182 to −3151 (distal) and −176 to −149 (proximal) show homology to the consensus p53-binding site. Using promoter deletion studies, we find that both proximal and distal elements are required for high level, synergistic transcriptional activation in vivo. Electron microscopy indicates that p53-p53 interactions link proximal and distal p53-binding elements and cause looping out of intervening DNA, suggesting that this DNA sequence may be dispensable for synergy. This idea was confirmed by progressive deletion of the DNA between p53-binding elements. Synergism persisted with spacing reduced to only 150 bp. Tetramerization-deficient p53 mutants were defective for transcriptional activation but still capable of synergy. Our results provide evidence for a model by which high level transcriptional activation of promoters with multiple p53 response elements is achieved.

The HMG‐box mitochondrial transcription factor xl‐mtTFA binds DNA as a tetramer to activate bidirectional transcription

The mitochondrial HMG‐box transcription factor xl‐mtTFA activates bidirectional transcription by binding to a site separating two core promoters in Xenopus laevis mitochondrial DNA (mtDNA). Three independent approaches were used to study the higher order structure of xl‐mtTFA binding to this site. First, co‐immunoprecipitation of differentially tagged recombinant mtTFA derivatives established that the protein exists as a multimer. Second, in vitro chemical cross‐linking experiments provided evidence of cross‐linked dimers, trimers and tetramers of xl‐mtTFA. Finally, high resolution scanning transmission electron microscopy (STEM) established that xl‐mtTFA binds to the specific promoter‐proximal site predominantly as a tetramer. Computer analysis of several previously characterized binding sites for xl‐mtTFA revealed a fine structure consisting of two half‐sites in a symmetrical orientation. The predominant sequence of this dyad symmetry motif shows homology to binding sites of sequence‐specific HMG‐box‐containing proteins such as Sry and Lef‐1. We suggest that bidirectional activation of transcription results from the fact that binding of a tetramer of xl‐mtTFA permits symmetrical interactions with other components of the transcription machinery at the adjacent core promoters.

Structures of large T antigen at the origin of SV40 DNA replication by atomic force microscopy

For inorganic crystals such as calcite (CaCO3), Atomic Force Microscopy (AFM) has provided surface structure at atomic resolution (Ohnesorge and Binnig, 1993). As part of a broad effort to obtain high resolution for an individual protein or protein assembly (Binnig et al., 1986; Rugar and Hansma, 1990; Radmacher et al., 1992), we applied AFM to study the ATP-dependent double hexamer of SV40 large T antigen, which assembles around the viral origin of DNA replication. Multimeric mass has been determined in two-dimensional projected images by Scanning Transmission Electron Microscopy (STEM) (Mastrangelo et al., 1989). By AFM, if the DNA-protein preparation has been stained positively by uranyl acetate, the contour at the junction between hexamers is visible as a cleft, 2-4 nm deep. The cleft, whether determined as a fraction of height by AFM or as a fraction of mass thickness by STEM, is of comparable magnitude. On either side of the cleft, hexamers attain a maximum height of 13-16 nm. Monomers found in the absence of ATP show heights of 5-7 nm. Taken together, the z coordinates provide a surface profile of complete and partial replication assemblies consistent with the spatial distribution of recognition pentanucleotides on the DNA, and they contribute direct geometrical evidence for a ring-like hexamer structure.

Selection and differentiation of aminopterin resistant cells of Datura innoxia

Abstract Primary cultured pith cells of Datura innoxia were subjected to a stepwise selection of colonies resistant to the folic acid analog, aminopterin. The first two screenings were done at a concentration of 1 · 10−5 M, followed by 2 · 10−5 M and 4 · 10−5 M. The frequency of resistant colonies ranged from 5.4 · 10−6 to 10−7 during these steps. Resistance was maintained in cells through two subcultures (3 months) without aminopterin. Independently isolated resistant lines differed with respect to growth and ability to differentiate. Plants have been obtained from five resistant lines. One of these plants is fertile and has produced seeds.