To study the relationship between the primary structure of the c-myc protein and some of its functional properties, we made in-frame insertion and deletion mutants of the normal human c-myc coding domain that was expressed from a retroviral promoter-enhancer. We assessed the effects of these mutations on the ability of c-myc protein to cotransform normal rat embryo cells with a mutant ras gene, induce foci in a Rat-l-derived cell line (Rat-la), and localize in nuclei. Using the cotransformation assay, we found two regions of the protein (amino acids 105 to 143 and 321 to 439) where integrity was critical: one region (amino acids 1 to 104) that tolerated insertion and small deletion mutations, but not large deletions, and another region (amino acids 144 to 320) that was largely dispensable. Comparison with regions that were important for transformation of Rat-la cells revealed that some are essential for both activities, but others are important for only one or the other, suggesting that the two assays require different properties of the c-myc protein. Deletion of each of three regions of the c-myc protein (amino acids 106 to 143, 320 to 368, and 370 to 412) resulted in partial cytoplasmic localization, as determined by immunofluorescence or immunoprecipitation following subcellular fractionation. Some abnormally located proteins retained transforming activity; most proteins lacking transforming activity appeared to be normally located.

An important point of regulation in the reproductive growth and latency of the human and simian immunodeficiency viruses (HIV and SIV, respectively) is provided by virally encoded trans-activators (tat), proteins capable of dramaticaily increasing viral gene expression. The mechanism of this autostimulatory pathway has remained unclear, however, with substantial effects having been reported at the level of either niRNA accumulation, translational efficiency, or both. Our previous findings indicated that trans-activation results primarily from induction of RNA levels but could not distinguish between the roles of transcriptional rate, RNA stabilization, and RNA transport in this event. In addition, the boundaries of tat-responding elements, which would be valuable in elucidating the mode of tat action, are not precisely known. In this study, HIV-1 and HIV-2 long terminal repeat-directed expression was characterized by using an in vitro nuclear transcription assay to clarify this mechanism, and a detailed mutational analysis was undertaken to localize precisely the sequences participating in this process. Two key findings were revealed: an increased transcription rate was the primary event in tat-mediated activation of HIV-1 and HIV-2, and trans-activation was impaired by mutations in two regions, the TATA box and sequences between + 19 to +42, a region lacking enhancer activity. These results implicate a discrete 3' regulatory element in the transcriptional activation of the HIVs.

No abstract

No abstract

Transforming growth factor alpha (TGF-ot) is a transformation-responsive mitogenic polypeptide that is expressed in the brain, epithelial cells, and activated macrophages. We isolated and characterized the TGF-a promoter and localized the 5' end of the TGF-ea transcript to a unique position. Surprisingly, no apparent TATA box was present in the promoter sequence, suggesting that transcription from mammalian genes can initiate at unique and specific positions from promoters lacking this sequence motif.The transforming growth factors, which were first purified from retrovirally transformed cells, induce a reversible phenotypic transformation of some normal mammalian cells in culture (for reviews, see references 7 and 10). These factors have been classified into two types based on their biological and physical properties: transforming growth factor alpha (TGF-a), which in its fully processed form is a 50-amino-acid peptide sharing about 30% sequence homology with epidermal growth factor (EGF), and TGF-,1l (a prototype for a family of proteins [30]), which is a structurally unrelated homodimer of 112 amino acids. The synthesis of TGF-a has been detected in a variety of transformed cell lines and tumors (8, 10), prompting suggestions that the protein plays a role in the growth of some tumors through autocrine or paracrine mechanisms (29). However, recent studies with cDNAs encoding both human (9) and rat (21) TGF-a have led to the detection of this gene not only in tumor tissue but also in normal adult (5,18,23,33) and embryonic (13,20,31,32) cells. As a first step in dissecting the genetic requirements for expression of this gene, we localized the 5' end of the endogenous human mRNA to a single site of transcription initiation. We isolated genomic DNA 5' of this gene and identified by transient assays a 300-base-pair (bp) fragment within the flanking region that exhibits bidirectional promoter activity. Sequence analysis of this upstream region revealed that despite the identification of potential binding sites for several transcription factors, no canonical CCAAT or TATA sequences were apparent, suggesting that transcription from mammalian genes can initiate at unique and specific positions from promoters lacking this sequence motif.Analysis of the 5' end of TGF-a mRNA. To define the 3' boundary of the TGF-a promoter, we localized the 5' end of human TGF-a mRNA by primer extension analysis. For this purpose we used a 25-mer oligonucleotide complementary to sequences immediately downstream of the TGF-a initiator ATG, spanning positions +4 to +28 which were defined relative to the ATG codon. Validation of the specificity of * Corresponding author. the oligomer for TGF-a was first provided by primer extension analysis of total cytoplasmic RNA isolated from cells that were stably transfected with a vector containing TGF-a cDNA under transcriptional control of the simian virus 40 (SV40) early promoter (28). Three extension products with lengths of 160, 155, and 105 nucleotides were identified (Fig.