Upstream sequences required for efficient expression of a soybean heat shock gene.

Abstract

A soybean gene (Gmhspl7.5-E) encoding a small heat shock protein was introduced into primary sunflower tumors via T-DNA-mediated transformation. RNA blot hybridizations and Sl-nuclease hybrid protection studies indicated that the heat shock gene containing 3.25 kilobases of 5'-flanking sequences was strongly transcribed in a thermoinducible (40°C) manner. Transcriptional induction also occurred to a lesser extent upon treatment of whole tumors with sodium arsenite and CdCl2. Basal (26°C) transcription was not detected in soybean seedlings, but it was quite evident in transformed tumor tissue. A 5' deletion to -1,175 base pairs with respect to the CAP site had no effect on the levels of thermoinducible transcription, but it resulted in a large increase in basal transcription. Further removal of DNA sequences (including the TATA-distal heat shock consensus element) to -95 base pairs reduced thermoinducible transcription by 95% and also greatly decreased basal transcription. The termini of the Gmhspl7.5-E RNA in the tumor were generally the same as those present in soybean RNA, with the exception of several additional 3' termini.The exposure of higher plants to elevated temperatures elicits a strong response at both the translational (1,4,5,12,20,24) and transcriptional (14, 35) levels. The heat shock (HS) response of plants is in general similar to that observed in many higher organisms including Drosophila melanogaster, in which it is best characterized (2). In soybean plants, thermal stress results in the rapid loss of polysomes and in a decreased synthesis of many normally abundant proteins (24). A new set of RNAs also rapidly appears which is translated into several classes of HS-specific proteins (22,23,35). The low-molecular-weight group is predominantly expressed and contains from 30 to 50 members (20,24). This complex group of low-molecular-weight heat shock proteins (HSPs) shows a similarity in hydropathy profiles and amino acid sequences (15,21,29,36) to the small HSPs of D. melanogaster (39), Xenopus laevis (7), and Caenorhabditis elegans (33).In addition to similarities in the protein structure of HSPs among diverse groups of eucaryotes, the regulatory mechanisms responsible for thermally inducible transcription also appear to be highly conserved. A direct demonstration of the universal aspects in HS promoter design and function is provided by examples of heterologous expression of the Drosophila HSP70 promoter in mammalian (10,13,28,32) and amphibian cells (44). Deletion studies (31) and the insertion of synthetic DNA oligomers (32) identified a region 13 to 28 bases upstream from the TATA motif that is essential for the thermoinducibility of Drosophila HSP70 transcription. In Drosophila HS genes, this TATA-proximal element forms the consensus sequence CT-GAA--TTC-AG (31) and lies within a region that interacts with a HS-specific transcription factor (30,46 upstream from the TATA-like region in several soybean HS genes (15,36).This study represents the first step in the definition and characterization...

Functional domains of a T-DNA promoter active in crown gall tumors.

“…Large Grey) seedlings, and the plants were grown as previously described (20). After 14 to 16 days of tumor growth, an average of 200 to 300 tumors for each promoter mutation were harvested and immediately frozen with liquid nitrogen.…”

“…Transcript levels of both the 780 test gene and reference gene were assayed by S1 nuclease hybrid protection. Analyses were performed with approximately 15 ,ug of poly(A)-enriched RNA isolated from sunflower tumors as previously described (10,20). The hybridization probe (+60 to -74 bp; see Fig.…”

Role of the Leader Sequence during Thermal Repression of Translation in Maize, Tobacco, and Carrot Protoplasts

“…The transcriptional regulation of heat-shock genes has been highly conserved throughout eukaryotes, including plants (9,16,18,19).…”

“…As a consequence of thermal stress, the transcription of heat-shock genes, which were previously either silent or expressed at low levels, is increased both dramatically and rapidly, whereas other genes are transcriptionally repressed. Moreover, heat-shock messages are preferentially translated over the preexisting cellular mRNAs at elevated temperatures (12).The transcriptional regulation of heat-shock genes has been highly conserved throughout eukaryotes, including plants (9,16,18,19). …”

“…The transcriptional regulation of heat-shock genes has been highly conserved throughout eukaryotes, including plants (9,16,18,19). All heat-shock genes investigated to date contain several copies of a 14-bp HSE upstream of the TATA box of the promoter that was first described for the Drosophila hsp70 ' 4Abbreviations: hsp, heat-shock protein; HSE, heat-shock element; CAT, chloramphenicol acetyltransferase; GUS, ,B-glucuronidase; Luc, luciferase; NOS, nopaline synthase polyadenylation site; TMV, tobacco mosaic virus.…”