No abstract

Max is a basic helix-loop-helix/leucine zipper protein that forms heterodimers with the Myc family of proteins to promote cell growth and with the Mad/Mxi1 family of proteins to inhibit cell growth. The role of Max as the obligate binding partner for these two protein families necessitates the observed constitutive expression and relatively long half-life of the max mRNA under a variety of growth conditions. In this study, we have used the chicken max gene to map DNA elements maintaining max gene expression in vertebrate cells. We have identified a minimal regulatory region (MRR) that resides within 115 bp of the max translation initiation site and that possesses an overall structure typical of TATA-less promoters. Within the MRR are two consensus binding sites for Sp1, a ubiquitously expressed transcription factor that plays a role in the expression of many constitutive genes. Interestingly, we show that direct binding by Sp1 to these sites is not required for MRR-mediated transcription. Instead, the integrity of a 20-bp DNA element in the MRR is required for transcriptional activity, as is the interaction of this DNA element with a 90-kDa cellular protein.Our data suggest that it is the persistence of this 90-kDa protein in vertebrate cells which drives max gene expression, insulates the max promoter from the dramatic changes in transcription that accompany cell growth and development, and ensures that adequate levels of Max will be available to facilitate the function of the Myc, Mad, and Mxi1 families of proteins.Max is a nuclear phosphoprotein of the basic helix-loophelix/leucine zipper (bHLH/LZ) class of transcription factors (13,42,55,62). The Max protein associates with the Myc family of oncoproteins in vitro and in vivo (1-3, 12, 13, 42, 51, 54, 62, 63) and forms heterodimers which bind to a core consensus DNA sequence, CACGTG, referred to as the Myc E box (3,12,13,42,63). Myc-Max heterodimer formation has been shown to be essential for all of the known biological functions of Myc, including the induction of cell growth (1), the triggering of apoptosis under specific growth conditions (1), cellular transformation (2, 54, 62), and the regulation of target gene transcription (3,27,59). Several groups have observed a dual effect of Max on Myc function, with an increase in Mycmediated cellular transformation noted when Max levels are elevated modestly and a decrease in transformation when Max levels are extremely high (2, 57, 61). Since Max does not possess a transcription activation domain (42, 55) and forms homodimers that bind to the same target DNA sites as MycMax heterodimers (10-12, 42, 51, 62), it has been proposed that a small increase in Max provides the protein necessary to maximize Myc-Max activity, while high Max levels promote the formation of inactive Max-Max homodimers (4,32,57,63). With the recent discovery of the Mad (7, 36) and Mxi1 (77) proteins-additional bHLH/LZ proteins that lack transcription activation domains and preferentially dimerize with Max (not Myc) to bind to Myc E-box DNA ...

One isoform of maize glutamine synthetase, encoded by GS1‐2, is localized exclusively in the maternal tissues of the developing kernel. Previously, we have demonstrated the ability of the proximal 664 base pair 5′ upstream portion of GS1‐2 to drive maternal tissue‐specific GUS expression in transgenic maize kernels (Muhitch et al. Plant Sci. 163: 865–872). In this report, a series of GS1‐2 promoter/GUS reporter transgenes, progressively truncated from the 5′ end of the full length 664 base pair promoter, were evaluated for transformation efficiency and their ability to drive tissue‐specific gene expression in transgenic maize. Analysis of transgene integration and expression suggests that GS1‐2/GUS transgenes were incorporated efficiently into the maize genome, but were not expressed efficiently in maize cells. Truncation of the promoter from −664 to −394, −206 or −72, relative to the putative transcription start site, resulted in the loss of tissue specific expression within the kernels of transformed plants. Among the truncated series, moderate staining was exhibited by the −394 promoter/GUS gene transformants, stronger staining was found in −206 promoter/GUS gene transformants, but relatively weak and variable staining occurred in plants transformed with the −72/GUS gene. Likely explanations for these observations are considered.