Positive and negative cis-acting DNA domains are required for spatial and temporal regulation of gene expression by a seed storage protein promoter.

Mutations affecting spatial and temporal regulation of a beta-phaseolin gene encoding the major storage protein of bean (Phaseolus vulgaris) were analyzed by stable and transient transformation approaches. The results substantiate the value of transient assays for rapid determination of the functionality of cis-acting sequences and the importance of stable transformation to identify tissue-specific determinants. Spatial information is specified primarily by two upstream activating sequences (UAS). UAS1 (-295 to -109) was sufficient for seed-specific expression from both homologous and heterologous (CaMV 35S) promoters. In situ localization of GUS expression in tobacco embryos demonstrated that UAS1 activity was restricted to the cotyledons and shoot meristem. A second positive domain, UAS2 (-468 to -391), extended gene activity to the hypocotyl. Temporal control of GUS expression was found to involve two negative regulatory sequences, NRS1 (-391 to -295) and NRS2 (-518 to -418), as well as the positive domain UAS1. The deletion of either negative element caused premature onset of GUS expression. These findings indicate combinatorial interactions between multiple sequence motifs specifying spatial information, and provide the first example of the involvement of negative elements in the temporal control of gene expression in higher plants.     

A putative ancestral actin gene present in a thermophilic eukaryote: novel combination of intron positions.

The gene encoding actin in the thermophilic fungus Thermomyces lanuginosus has been isolated and sequenced. It contains five introns, with three being at positions already known to be intron sites in actin genes from other eukaryotes. These three positions have not been found to occur simultaneously in any other organisms to date, suggesting that the actin gene in this fungus may more closely resemble an ancestral form of this highly conserved eukaryotic gene. The 5' flanking region of the gene contains a TATA-like sequence and two CCAAT motifs in positions almost identical to those in the yeast actin gene. Other features of the gene sequence, and possible adaptations to thermophily, are discussed.     

Mutational analysis of upstream activation sequence 2 of the CYC1 gene of Saccharomyces cerevisiae: a HAP2-HAP3-responsive site.

We analyzed upstream activation sequence 2 (UAS2), one of two independent UAS elements in the CYC1 gene of Saccharomyces cerevisiae. Deletions and linker scanning mutations across the 87 base pairs previously defined as UAS2 showed two separate functional elements required for full activity. Region 1, from -230 to -200, contains the principal activation site and responds to the trans-acting regulatory loci HAP2 and HAP3. A portion of region 1 is homologous to two other HAP2-HAP3-responsive UASs and includes the G----A transition mutation UP1, which increases UAS2 activity. This consensus sequence TNATTGGT bears striking similarity to several CAAT box sequences of higher cells. Region 2, from -192 to -178, substantially enhances the activity of region 1, yet has little activity by itself. These regions bind distinct proteins found in crudely fractionated yeast extracts.