ACGT and vicilin core sequences in a promoter domain required for seed-specific expression of a 2S storage protein gene are recognized by the opaque-2 regulatory protein

The expression of Brazil nut storage albumin genes is highly regulated during seed development. Several sequences in the promoter of one of these genes show homologies with the target sites of the maize O2 bZIP regulatory protein. We therefore asked whether the O2 protein would recognize these promoter sequences. We show that the O2 protein binds to three different sequences (F1, F2 and F3). F1 and F3 are hybrid C/G and A/G boxes, respectively, that are homologous to the O2-binding site of a maize -zein gene. F2 is a new O2-binding sequence related to the O2 target sites of the Coix -coxin, the maize b-32 genes and the AP-1 pseudopalindrome. Molecular modelling showed that an Asn and a Ser in the 02 DNA binding domain make different base-specific contacts with each operator. 5 Promoter deletions of the be2S1 gene showed that the domain containing the O2 target sites F1 and F2 is required for detectable reporter gene expression in transgenic tobacco seeds. Moreover, the homologous coix O2 protein was shown to in situ transactivate the promoter region encompassing the three O2-binding sites F1, F2 and F3. Thus, these sites may be in vivo regulatory sequences mediating activation by bZIP regulatory proteins.     

Positive and negative regulation of basal expression of a yeast HSP70 gene.

The SSA1 gene, one of the heat-inducible HSP70 genes in the yeast Saccharomyces cerevisiae, also displays a basal level of expression during logarithmic growth. Multiple sites related to the heat shock element (HSE) consensus sequence are present in the SSA1 promoter region (Slater and Craig, Mol. Cell. Biol. 7:1906-1916, 1987). One of the HSEs, HSE2, is important in the basal expression of SSA1 as well as in heat-inducible expression. A promoter containing a mutant HSE2 showed a fivefold-lower level of basal expression and altered kinetics of expression after heat shock. A series of deletion and point mutations led to identification of an upstream repression sequence (URS) which overlapped HSE2. A promoter containing a mutation in the URS showed an increased level of basal expression. A URS-binding activity was detected in yeast whole-cell extracts by a gel electrophoresis DNA-binding assay. The results reported in this paper indicate that basal expression of the SSA1 promoter is determined by both positive and negative elements and imply that the positively acting yeast heat shock factor HSF is responsible, at least in part, for the basal level of expression of SSA1.     

Identification of cis-acting DNA elements involved in the regulation of angiotensinogen gene expression.

Angiotensinogen is the precursor molecule of one of the most potent vasoactive substances, angiotensin-II. Angiotensinogen is normally synthesized in the liver and secreted into the plasma where it is converted into angiotensin-II by the combined proteolytic action of renin and angiotensin converting enzyme. Angiotensinogen levels in the plasma are modulated by a number of pathological and physiological factors. In order to understand the regulation of angiotensinogen gene expression, we have constructed an expression vector in which 688 bp of the 5'-flanking region of the rat angiotensinogen gene were attached to the chloramphenicol acetyl transferase (CAT) coding sequence. We have also obtained 5'-sequential deletion mutants from the rat angiotensinogen promoter attached to the CAT gene, and have identified multiple cis-acting DNA sequences involved in the regulation of angiotensinogen gene expression by transient transfection of these recombinant DNA molecules in human hepatoma cell lines, Hep3B, and HepG2.     

Four distinct nuclear proteins recognize in vitro the proximal promoter of the bean seed storage protein β-phaseolin gene conferring spatial and temporal control

A proximal promoter (-422/-13) of the bean seed storage protein beta-phaseolin gene contains cis-regulatory elements conferring spatial and temporal gene regulation. To correlate trans-acting elements with these cis-elements, we performed gel mobility shift and exonuclease III protection assays using bean seed nuclear proteins, and identified target sequences of four DNA-binding proteins associated with this promoter. Three CANNTG motifs, CACGTG (-248/-243), CACCTG (-163/-158), and CATATG (-100/-95), were determined as target sequences of the same DNA-binding protein designated CAN. Competition assays using oligonucleotides containing the wild-type or mutated CANNTG motif indicated that the CANNTG motif appears to be a preferred target sequence for CAN binding. Competition assays also demonstrated that DNA-binding protein AG-1 binds to AAAAAG(A/G)CAA (-356/-347, -191/-182), CA-1 binds to two CA-rich sequences (-201/-192, -175/-160), and that a TATA-box binding protein binds to either TATATAA (-43/-37) or TATAAA (-32/-27) or both. Based on these and other results, it is proposed that CACGTG motif (-248/-243) is a major cis-acting regulatory element conferring spatial and temporal control of the beta-phaseolin gene.     

Developmental and spatial regulation of a Dictyostelium prespore gene: cis-acting elements and a cAMP-induced, developmentally regulated DNA binding activity.

Previously, 5' deletion analysis revealed three important upstream regions within the regulatory region of the cAMP-induced, prespore gene SP60 of D. discoidium, each of which contains a CA-rich sequence element (CAE: consensus CACACAYYYCACACAAA/T). In this study, we have made site-directed mutations within these CAEs and examined their effect on reporter gene activity (luciferase or lacZ). Point mutations within or deletion of the distal CAE (CAE-1), middle CAE (CAE-2) or proximal CAE (CAE-3) result in substantial decreases in promoter activity at 18 h of development or in response to cAMP. lacZ fusions made with the CAE mutant promoters produced novel beta-gal staining patterns that suggest the presence of one or more morphogen gradients within the prespore zone of the slug and indicate that the CAEs are also important in regulating the spatial patterning of SP60 expression in the multicellular aggregate. Gel mobility shift assays were used to identify activities from crude nuclear extracts that bind oligonucleotides containing the CAEs. One of the binding activities is not observed in extracts from vegetative cells or cells in early development and is induced during multicellular development with kinetics similar to those of SP60 gene expression. This activity is also induced in response to cAMP and specifically binds the wild-type CAE-1- and CAE-2-containing oligonucleotides. CAE-1 and CAE-2 oligonucleotides containing point mutations within the CAE core sequence neither bind to nor compete for the cAMP-induced, developmentally regulated factor(s) and result in substantial reductions in expression levels when substituted for the wild-type CAEs in vivo. The correlation between in vitro binding and in vivo function suggests that the CAE-1/CAE-2 binding activity may be involved in regulating cAMP and developmentally induced expression of SP60. A second, constitutive in vitro binding activity with high affinity to CAE-3 is also described. Models are proposed to relate the binding activities with the effects of the mutations on the spatial patterning of SP60-lacZ expression.     

The murine lymphotoxin gene promoter. Characterization and negative regulation

Murine lymphotoxin (LT; TNF-beta) gene upstream regulatory elements were identified by linking fragments of 5' DNA to the chloramphenicol acetyl transferase gene. Fragment LT1 (-293 to +77 in relation to the proximal cap site) exhibited promoter activity which drove CAT expression in transfected murine fibroblasts and T lymphomas. Primer extension analysis of endogenous LT message confirmed that LT1 contained the necessary elements required for promoter function. Promoter activity was not observed when LT2 (-662 to +77), LT3 (-1186 to +77), or LT3 delta AX (-1186 to +77 (delta-662/-269)) were ligated to the chloramphenicol acetyl transferase gene and transfected into fibroblasts or T lymphomas. At least one upstream repressor element is postulated to account for this promoter inhibition. In contrast to the results obtained with fibroblast and T cell transfectants, LT1 was inactive in the B cell transfectants A20 and P3X63. This suggests that some B cells express a repressor factor that inhibits the LT promoter and/or they lack the necessary positive regulatory factors.