Structural and functional analyses of Arabidopsis thaliana chlorophyll a/b-binding protein (cab) promoters

Arabidopsis thaliana carries three functional copies of the chlorophyll a/b-binding protein (cab) gene which code for an identical mature protein. DNA sequence comparison of all three cab promoters indicated that cab2 and cab3 are more closely related compared to cab1. Although the highest degree of homology was found between the TATA box and -256 of cab3 promoter, suggesting that this region plays a major role in promoter function, this promoter regions are only 47% homologous. To study whether these promoters are regulated by identical cis-acting regulatory elements, the promoters were mutated by progressive deletions and the effects on the promoter activity were measured in either transformed plants or cultured cells. It was found that the minimum sequence necessary for the light-dependent tissue-specific promoter activity of the cab3 is the 89 bp DNA fragment (between -74 and -164) at the region of the TATA and the CCAAT boxes. However, an additional 45 bp DNA fragment (between -164 and -209) upstream of the CCAAT box was necessary for the full promoter activity in the leaves. The regulatory element in the 45 bp region appears to be a positive regulator or enhancer which is specific to photosynthetic cells, since the region did not enhance the promoter activity in cultured cells. This region contains an octamer, TGCCACGT (cab2) or TGCCACAT (cab3), which is similar to the previously identified element, TGACACGT from Arabidopsis cab1 promoter. The upstream regions of the cab promoters appear to contain additional elements which are functionally distinct in each promoter since the upstream region of cab1 activated a non-functional nos promoter whereas that of cab3 did not.     

Gene regulation by tyrosine kinases: src protein activates various promoters, including c-fos.

A promoter of the nuclear proto-oncogene fos was activated by cotransfection with the viral src gene. Ability to transactivate the c-fos promoter was dependent on tyrosine kinase activity, because (i) src mutants which have reduced tyrosine kinase activity due to mutation of Tyr-416 to Phe showed lower promoter activation, (ii) pp60c-src mutants which have increased tyrosine kinase activity due to mutation of Tyr-527 to Phe also augmented c-fos promoter induction, and (iii) mutation in the ATP-binding site of pp60v-src strongly suppressed c-fos promoter activation. Tyrosine kinase activity alone, however, was not sufficient for promoter activation, because of pp60v-src mutant which lacked its myristylation site and consequently membrane association showed no increased c-fos promoter activation. Both the tyrosine kinase- and membrane-association-defective mutants were also unable to induce transformation. Therefore, phosphorylation of membrane-associated substrates appears to be required for both gene expression and cellular transformation by the src protein. Two regions of the c-fos promoter located between positions -362 and -324 and positions -323 and -294 were responsive to src stimulation. We believe that protein tyrosine phosphorylation represents an important step of signal transduction from the membrane to the nucleus.     

Construction of strong mammalian promoters by random cis-acting element elongation

Synthetic oligonucleotides containing one of four kinds of cis-acting elements, binding sites for activating protein-1 (AP-1), nuclear factor kappaB (NF-kappaB), CArG binding factor A (CBF-A), and nuclear factor Y (NF-Y), were randomly ligated to construct DNA fragments. These fragments were inserted into the SalI site of a promoter probe vector; pGL3-TATASal, which is located immediately upstream of the TATA box sequence of the human heme oxygenase 1 gene and linked to the luciferase gene to construct 11 plasmid vectors. When these vectors were introduced into PC-3 cells of human prostate cancer, 6 out of the 11 transfectants showed a significantly higher luciferase activity than pGL3-TATASal. The two strongest promoters (clone 6 and clone 11) were investigated further Clone 6 turned out to be the strongest, showing a 3.0- and 8.4-fold activity in comparison to the two frequently used promoters--the cytomegalovirus (CMV) immediate early promoter and the simian virus 40 (SV40) early promoter respectively. Clone 11 was less active than clone 6, but still showed higher activity than the two promoters. When the plasmids were introduced into nine other cell lines, their activities varied but were still comparable to the two promoters. These results indicate that the method used here is simple and efficient for constructing strong promoters that are potentially useful for vectors in either gene therapy or recombinant vaccine.