The 5''-proximal region of the wheat Cab-1 gene contains a 268-bp enhancer-like sequence for phytochrome response.

We have previously reported that the expression of the wheat Cab-1 gene is subject to phytochrome regulation and a 1.8-kb 5' upstream sequence of this gene is sufficient for the regulated expression. To delineate sequences for the phytochrome response we analyzed a series of 5' deletion mutants as well as chimeric gene constructs comprising different sequences of the Cab-1 upstream region in transgenic tobacco seedlings. We found that a deletion mutant containing a 357-bp 5' upstream sequence still exhibits maximal levels of phytochrome-regulated expression. A 268-bp enhancer-like element, located between -89 and -357, is responsible for the phytochrome response of the Cab-1 gene; sequences upstream from -357 to -843 and downstream from -124 to +1100 are probably not involved. Finally, we show that the Cab-1 mRNA stability is not regulated by phytochrome.     

Multiple copies of a G-rich element upstream of a cAMP-inducible Dictyostelium gene are necessary but not sufficient for efficient gene expression.

The cysteine proteinase 1 (CP1) and cysteine proteinase 2 (CP2) genes of Dictyostelium discoideum encode co-ordinately expressed mRNA sequences which are inducible by extracellular cAMP. There are short, G-rich sequence elements upstream of both genes and we have previously shown that deletion of these elements from the CP2 gene abolishes cAMP-inducibility. We show here that the G-rich element from the CP1 gene is functionally homologous to that in the CP2 gene by reconstituting cAMP-inducibility in a deletion mutant of the CP2 gene using CP1-derived sequences. Both the CP1 and CP2 genes contain multiple G-rich elements. We show that efficient induction requires at least two copies of the CP1 element and that their relative orientation is unimportant. Two copies of an inverted relative orientation are, however, inactive when moved upstream of their normal position and are incapable of conferring cAMP-inducibility on a heterologous gene. These observations suggest that these sequences are either essential promoter elements, not themselves interacting with the inducer, or that their interaction with a separate class of control sequences is necessary for inducible expression.     

EVX2, a human homeobox gene homologous to the even-skipped segmentation gene, is localized at the 5' end of HOX4 locus on chromosome 2.

We isolated and mapped three new human homeoboxes located on chromosome 2 upstream from the reported seven HOX4 homeobox sequences. Two of them, HOX41 and HOX4H, clearly belong to the HOX gene family, in particular to homology groups 1 and 2, and possibly represent the most 5' HOX4 homeoboxes. A third homeobox 13 kb upstream from HOX41 was identified. Sequencing data show that this is the human homolog of the murine Evx-2 homeobox. Both homeoboxes are closely related to the murine Evx-1 and to the frog Xhox-3 homeoboxes. The four genes represent vertebrate homologs of Drosophila even-skipped (eve), a segmentation gene of the pair-rule class. Human EVX2 sequences belong to an active gene because they are transcribed and properly processed in cells and tissues. We have identified for the first time a homeogene of a different class at a HOX locus. These findings are relevant to the understanding of the evolution of HOX gene clusters and their regulation.     

Identification and functional validation of a 5' upstream regulatory sequence in the human tyrosinase gene homologous to the locus control region of the mouse tyrosinase gene

Comparison analysis of the sequences of the mouse and human genomes has proven a powerful approach in identifying functional regulatory elements within the non-coding regions that are conserved through evolution between homologous mammalian loci. Here, we applied computational analysis to identify regions of homology in the 5' upstream sequences of the human tyrosinase gene, similar to the locus control region (LCR) of the mouse tyrosinase gene, located at -15 kb. We detected several stretches of homology within the first 30 kb 5' tyrosinase gene upstream sequences of both species that include the proximal promoter sequences, the genomic region surrounding the mouse LCR, and further upstream segments. We cloned and sequenced a 5' upstream regulatory sequence found between -8 and -10 kb of the human tyrosinase locus (termed h5'URS) homologous to the mouse LCR sequences, and confirmed the presence of putative binding sites at -9 kb, homologous to those described in the mouse tyrosinase LCR core. Finally, we functionally validated the presence of a tissue-specific enhancer in the h5'URS by transient transfection analysis in human and mouse cells, as compared with homologous DNA sequences from the mouse tyrosinase locus. Future experiments in cells and transgenic animals will help us to understand the in vivo relevance of this newly described h5'URS sequence as a potentially important regulatory element for the correct expression of the human tyrosinase gene.     

Efficiently finding regulatory elements using correlation with gene expression

We present an efficient algorithm for detecting putative regulatory elements in the upstream DNA sequences of genes, using gene expression information obtained from microarray experiments. Based on a generalized suffix tree, our algorithm looks for motif patterns whose appearance in the upstream region is most correlated with the expression levels of the genes. We are able to find the optimal pattern, in time linear in the total length of the upstream sequences. We implement and apply our algorithm to publicly available microarray gene expression data, and show that our method is able to discover biologically significant motifs, including various motifs which have been reported previously using the same data set. We further discuss applications for which the efficiency of the method is essential, as well as possible extensions to our algorithm.     

Insertion of (dA-dT)n sequences into the regulatory region of the pho5 gene inhibits its expression

We have studied the effect of some regular sequences namely (dA-dT)n, (dA)n and (dG-dC)n on the Saccharomyces cerevisiae PHO5 gene expression. These sequences were inserted into the ClaI site, located between two phosphate-responsible upstream activating sequences (UAS's). A modified PHO5 gene cloned in vector plasmids was used to transform the pho3, pho5 yeast strain and the level of acid phosphatase expression in various conditions was measured. We show that the insertion of (dA-dT)n blocks, but not (dA)n or (dG-dC)n, leads to the dramatic decrease of PHO5 gene expression in a derepressed state. (dA-dT)n inserts also inhibit the expression of PHO5 gene which was put under the control of heterologous UASgal.     

Multiple domains exist within the upstream activator sequence of the octopine synthase gene.

It is known that a 16-base pair palindrome (ACGTAAGCGCTTACGT) located upstream of the ocs gene can activate a maize adh1 promoter in a transient expression system [Ellis et al. (1987). EMBO J. 6, 11-16; Ellis et al. (1987). EMBO J. 6, 3203-3208]. We have determined that this palindrome is also essential for ocs promoter activity in tobacco calli. In addition, sequences immediately adjacent to this palindrome, both 5' and 3', modulate its activity. The palindrome is sensitive to the differentiated state of the plant cells in which it resides; it is active in calli and the leaves of small shoots but is inactive in the leaves of rooted plants. We have tested upstream sequences from two other T-DNA genes that have homology to this palindrome for their ability to activate the octopine synthase promoter in tobacco calli. The upstream region from the mannopine synthase gene can activate the octopine synthase promoter, but an upstream region from the gene implicated in octopine and nopaline secretion cannot activate the promoter.