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Templates constructed from the wheat Em and maize rab28 promoters are efficiently and accurately transcribed in the well-characterized cell-free transcription system prepared from HeLa nuclei. Deletion analysis of the Em promoter indicates that a G-box (CACGTG) element (Em1 b) is required for transcription. USF, a Myc transcription factor in HeLa nuclear extracts, activates transcription by binding t o Emlb, as shown by the ability of an antibody raised against USF t o inhibit transcription and t o interfere with Em1 b complex formation in an electrophoretic mobility shift assay. The addition of the recombinant Viviparousl protein from maize t o HeLa nuclear extracts specifically stimulated transcription of the Em promoter but was dependent on the presence of USF in the extract. In USF-depleted extracts, the addition of recombinant EmBP1, a basic leucine zipper transcription factor from wheat, activated transcription through Em1 b as well as from a similar G-box in the adenovirus major late promoter. Our study demonstrates that the basic transcriptional apparatus in HeLa nuclear extract supports transcription from plant promoters and can be used t o assay the function of certain plant nuclear proteins, thereby helping t o determine their effects on transcription.

In spite of their critical importance in myocardial hypertrophy and benign prostatic hyperplasia, nothing is known about mechanisms underlying transcriptional regulation of alpha 1a-adrenergic receptors (alpha 1aARs). Therefore we cloned 6.2 kb of novel sequence upstream of the initiator ATG in the human alpha 1aAR gene. Sequence analysis reveals a TATA-less promoter, the presence of several initiator (Inr) consensus sequences, multiple GC rich regions consistent with Sp-1 binding, and consensus sequences for AP-1 and AP-2 as well as putative cis transcriptional regulatory elements for binding of CREB (cyclic-AMP response element binding protein), glucocorticoids, estrogen, and insulin. Compared to the alpha 1bAR, the alpha 1aAR has several more cis regulatory elements, suggesting more complex regulation. The importance of alpha 1aARs in human disease makes it imperative to determine mechanisms underlying transcription and ultimately expression of this receptor. These studies can now be undertaken with the availability of human alpha 1aAR 5'-flanking and 5'-untranslated sequence.

Templates constructed from the wheat Em and maize rab28 promoters are efficiently and accurately transcribed in the well-characterized cell-free transcription system prepared from HeLa nuclei. Deletion analysis of the Em promoter indicates that a G-box (CACGTG) element (Em1b) is required for transcription. USF, a Myc transcription factor in HeLa nuclear extracts, activates transcription by binding to Em1b, as shown by the ability of an antibody raised against USF to inhibit transcription and to interfere with Em1b complex formation in an electrophoretic mobility shift assay. The addition of the recombinant Viviparous1 protein from maize to HeLa nuclear extracts specifically stimulated transcription of the Em promoter but was dependent on the presence of USF in the extract. In USF-depleted extracts, the addition of recombinant EmBP1, a basic leucine zipper transcription factor from wheat, activated transcription through Em1b as well as from a similar G-box in the adenovirus major late promoter. Our study demonstrates that the basic transcriptional apparatus in HeLa nuclear extract supports transcription from plant promoters and can be used to assay the function of certain plant nuclear proteins, thereby helping to determine their effects on transcription.

Conditions for transcription and nucleosome assembly of plasmids bearing Xenopus 5S RNA genes have been monitored in the whole oocyte S-150 extract (1). We find that the optimal conditions for transcription differ substantially from optimal conditions for nucleosome assembly. DNA molecules bearing as few as 50% of the native density of nucleosomes are transcriptionally inert. Although the 5S gene-specific transcription factor TFIIIA is in excess in this extract, these nucleosome reconstitutes do not exhibit TFIIA-like DNase footprints nor do these reconstitutes bind exogenous TFIIIA. We have also examined the nucleotide requirement for DNA supercoiling and for generation of 5S gene transcription complexes. Supercoiling associated with nucleosome assembly does not require ATP; however, nucleotide hydrolysis is required for establishment of active complexes. Phosphorylation of a 200 kdalton protein occurs in a 5S DNA-dependent manner concurrent with the generation of primed transcription complexes. Results of nondenaturing gel electrophoresis coupled with a second dimension of SDS gel electrophoresis suggest that the 200 kD protein may be a component of the 5S RNA gene transcription complex.