Identification and Characterization of a Bidirectional Promoter from the Intergenic Region between the Human DDX13 and RD Genes

The human DDX13 gene encodes a putative RNA helicase of the DExH-box family. In an earlier report we showed that the human DDX13 and RD genes were arranged head-to-head in the class III MHC complex and their ATG start codons were separated by 745 base pairs. We have now analyzed the common 745 bp intergenic region in detail and characterized their promoters. Northern blot analysis revealed that DDX13 and RD exhibit distinct patterns of steady-state expression among multiple human tissues. The promoter regions for DDX13 and RD genes were identified by deletion analysis from 740 bp to 176 bp of the intergenic region fused to a chloramphenicol acetyltransferase (CAT) reporter gene using transient transfection assays. Results indicated that a promoter sequence as small as 176 bp is sufficient for basal expression of both genes in HeLa and HepG2 cells. Functional analysis using a bidirectional reporter system demonstrates that the sequence 262 bp proximal to the DDX13 gene is sufficient for concurrent expression in both directions. However, the common 740 bp intergenic region showed promoter activity in DDX13 only, suggesting the presence of a negatively acting region for the RD gene within the region -267 to -744. It appears that RD expression is controlled by a complex system of positively and negatively acting elements present on distant portions of both genes.     

Two functional estrogen response elements are located upstream of the major chicken vitellogenin gene.

We used a transient-expression assay to identify two estrogen response elements (EREs) associated with the major chicken vitellogenin gene (VTGII). Each element was characterized by its ability to confer estrogen responsiveness when cloned in either orientation next to a chimeric reporter gene consisting of the herpes simplex virus thymidine kinase promoter and the chloramphenicol acetyl transferase-coding region. Deletion analyses indicated that sequences necessary for the distal ERE resided within the region from -626 to -613 (nucleotide positions relative to the VTGII start site) whereas those necessary for the proximal ERE were within the region from -358 to -335. These distal and proximal elements contain, respectively, a perfect copy and an imperfect copy of the 13-base-pair sequence that is an essential feature of the EREs associated with two frog vitellogenin genes. These chicken VTGII EREs mapped near regions that were restructured at the chromatin level when the endogenous VTGII gene was expressed in the liver in response to estradiol. These data suggest a model for the tissue-specific expression of this estrogen-responsive gene.     

Comparative Structural and Functional Characterization of Sorghum and Maize Duplications Containing Orthologous Myb Transcription Regulators of 3-Deoxyflavonoid Biosynthesis

Sequence characterization of the genomic region of sorghum yellow seed 1 shows the presence of two genes that are arranged in a head to tail orientation. The two duplicated gene copies, y1 and y2 are separated by a 9.084 kbp intergenic region, which is largely composed of highly repetitive sequences. The y1 is the functional copy, while the y2 may represent a pseudogene; there are several sequence indels and rearrangements within the putative coding region of y2. The y1 gene encodes a R2R3 type of Myb domain protein that regulates the expression of chalcone synthase, chalcone isomerase and dihydroflavonol reductase genes required for the biosynthesis of 3-deoxyflavonoids. Expression of y1 can be observed throughout the plant and it represents a combination of expression patterns produced by different alleles of the maize p1. Comparative sequence analysis within the coding regions and flanking sequences of y1, y2 and their maize and teosinte orthologs show local rearrangements and insertions that may have created modified regulatory regions. These micro-colinearity modifications possibly are responsible for differential patterns of expression in maize and sorghum floral and vegetative tissues. Phylogenetic analysis indicates that sorghum y1 and y2 sequences may have arisen by gene duplication mechanisms and represent an evolutionarily parallel event to the duplication of maize p2 and p1 genes.     

Molecular analysis of Arabidopsis endosperm and embryo promoter trap lines: reporter-gene expression can result from T-DNA insertions in antisense orientation, in introns and in intergenic regions, in addition to sense insertion at the 5' end of genes

Random insertions of promoterless reporter genes in genomes are a common tool for identifying marker lines with tissue-specific expression patterns. Such lines are assumed to reflect the activity of endogenous promoters and should facilitate the cloning of genes expressed in the corresponding tissues. To identify genes active in seed organs, plant DNA flanking T-DNA insertions (T-DNAs) have been cloned in 16 Arabidopsis thaliana GUS-reporter lines. T-DNAs were found in proximal promoter regions, 5' UTR or intron with GUS in the same (sense) orientation as the tagged gene, but contrary to expectations also in inverted orientation in the 5' end of genes or in intergenic regions. RT-PCR, northern analysis, and data on expression patterns of tagged genes, compared with the expression pattern of the reporter lines, suggest that the expression pattern of a reporter gene will reflect the pattern of a tagged gene when inserted in sense orientation in the 5' UTR or intron. When inserted in the promoter region, the reporter-gene expression patterns may be restricted compared with the endogenous gene. Among the trapped genes, the previously described nitrate transporter gene AtNRT1.1, the cyclophilin gene ROC3, and the histone deacetylase gene AtHD2C were found. Reporter-gene expression when positioned in antisense orientation, for example, in the SLEEPY1 gene, is indicative of antisense expression of the tagged gene. For T-DNAs found in intergenic regions, it is suggested that the reporter gene is transcribed from cryptic promoters or promoters of as yet unannotated genes.     

Regulation of Aspergillus nidulans penicillin biosynthesis and penicillin biosynthesis genes acvA and ipnA by glucose.

Expression of the Aspergillus nidulans penicillin biosynthesis genes acvA and ipnA, encoding delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase and isopenicillin N synthetase, respectively, was analyzed. The intergenic region carrying the divergently oriented promoters was fused in frame in both orientations to Escherichia coli lacZ and E. coli uidA reporter genes. Each construct permits simultaneous expression studies of both genes. Transformants of A. nidulans carrying a single copy of either plasmid integrated at the chromosomal argB locus were selected for further investigations. Expression of both genes was directed by the 872-bp intergenic region. ipnA- and acvA-derived gene fusions were expressed from this region at different levels. ipnA had significantly higher expression than did acvA. Glucose specifically reduced the production of penicillin and significantly repressed the expression of ipnA but not of acvA gene fusions. The specific activities of isopenicillin N synthetase, the gene product of ipnA, and acyl coenzyme A:6-aminopenicillanic acid acyltransferase were also reduced in glucose-grown cultures.