Characterization of the VirG binding site of Agrobacterium tumefaciens.

Expression of Agrobacterium tumefaciens virulence (vir) genes is dependent on the presence of a conserved 'vir box' sequence in their 5' nontranscribed regions. The location and number of these sequences vary considerably in different vir genes. Site-directed mutagenesis was used to identify the functional vir box(es) of virB, virC and virD. For virB expression both vir box B1 and B2 are required but only the vir box B1 is absolutely essential. Of the five vir boxes of virC and virD two are required for virC expression while only one vir box is required for virD expression. To investigate the minimum sequences necessary for vir gene induction a deletion derivative of virE that lacks the vir box region was used. This mutant is not induced by acetosyringone. The inducibility of this promoter was restored when a synthetic deoxyoligonucleotide dGTTTCAATTGAAAC was introduced at a location analogous to that of the wild type vir box sequence. Mutational analysis indicate that the functional vir box sequence is 14 residues in length, contains a dyad symmetry and has the consensus sequence d ryTncAaTTGnAaY [corrected] (r = purine, y = pyrimidine).     

Sequence and structure of the nucleolin promoter in rodents: characterization of a strikingly conserved CpG island

We report the isolation of the complete genes encoding nucleolin from rat and hamster. The DNA clones were obtained from partial genomic libraries by probing with a genomic DNA fragment containing the leader and promoter regions of the mouse nucleolin gene. We have determined the complete nucleotide sequence of the 5'-terminal region for the three rodent species. The sequenced regions extend over 1 kb downstream and upstream from the cap sites and include a conserved CpG island 1500 nucleotides (nt) long. The 5' end of the CpG island in each species has maintained a long alternating purine-pyrimidine sequence which could adopt a Z-DNA conformation. By sequence comparison, 42 blocks of homology are defined in the 5'-terminal region, of which 36 appear in the CpG island and contain numerous conserved CpG dinucleotides. Two blocks, 110 and 49 nt long, encompassing the cap sites and the region immediately upstream, respectively, present features characteristic of regulated genes: a possible TATA box (ATTA), two pyrimidine-rich nucleotide stretches and two inverted juxtaposed CCAAT-like boxes (GGTTGG). Furthermore, the adjacent upstream conserved region presents features characteristic of housekeeping genes: four G/C boxes, embedded in a high G + C-content sequence, among them one presenting a perfect consensus Sp 1-binding site (GCCCCGCCCC). Among unusual features, we report numerous large G + C-rich conserved sequences located in the first intron. One of these sequences contains two G/C boxes which border a sequence presenting a dyad symmetry (GCGCACGTGCTC). Our findings shed some light on the putative role of the CpG island. We show that CpG-rich sequence motifs are under strong selective pressure over the whole 5'-terminal region and are presumably involved in regulatory mechanisms.     

Sequences Distal to the AP1/E Box Motif Are Involved in the Cell Type-Specific Expression of the Rat Tyrosine Hydroxylase Gene

Abstract: In order to define cell type-specific elements associated with the catecholamine biosynthetic enzyme, tyrosine hydroxylase (TH), transient transfections of promoter deletion constructs were used to test relative reporter-gene activities in TH-expressing and-nonexpressing cell lines. Such assays demonstrated that a region between-503 and-578 contributed to rat TH promoter activity in the pheochromocytoma cell line PC12. Deletion of these sequences resulted in a 66% loss in cell type-specific activity. Mutations within the E box/dyad symmetry element (CAGGTGCCTGTGACAGTG) did not affect the basal and cell type-specific pattern of expression exhibited by the rat TH promoter. Promoter fusion constructs between the rat TH promoter (-741 and-197) and the human TH promoter (-197 and +1) exhibited reporter-gene activities equivalent to that of wild-type-741 rat TH constructs, further demonstrating that sequence elements upstream of the rat E box/dyad symmetry are important for cell type-specific expression. Gel-shift experiments indicated that a PC12 nuclear factor could bind to a 39-bp sequence within this region in a cell type-specific manner. The size of this factor was 52 kDa as determined by UV cross-linking experiments.     

Autoregulation of fos: the dyad symmetry element as the major target of repression.

Fos and Jun co-operatively repress the fos promoter. Removal of all putative Fos/Jun binding sites from the fos promoter neither obliterates the repression by Fos/Jun in transient cotransfection experiments in NIH3T3 cells nor the turn-off kinetics of serum-induced fos expression in stably transfected NIH3T3 cells. The dyad symmetry element (DSE) suffices to subject a promoter to this type of repression. However, one of the putative Fos/Jun binding sites (-292 to -299 and thus located immediately adjacent to the DSE), determines the very low level of basal expression.     

Characterization of the promoter elements for the staphylococcal enterotoxin D gene

Deletion analysis of the promoter for the Staphylococcus aureus enterotoxin D determinant indicated that a 52-bp sequence, from -34 to +18, was sufficient for sed promoter function and agr regulation. A consensus -10 Pribnow box sequence, a less conserved -35 sequence, and a TG dinucleotide motif were present. Transcribed sequences (+1 to +18) are essential for promoter activity.     

Molecular characterization of the vir regulon of Agrobacterium tumefaciens: complete nucleotide sequence and gene organization of the 28.63-kbp regulon cloned as a single unit

The entire vir regulon of Agrobacterium tumefaciens was subcloned and the complete 28.6-kbp nucleotide sequence was determined. The regulon was cloned as a single unit into two replicons, one of which replicates at a high copy number in this bacterium, and a second which has broad-host-range features to replicate in other Gram-negative bacteria. These vir region plasmids are able to confer in trans the processing and transfer activities on a second plasmid containing the T-DNA. In the high copy number vir region plasmid pUCD2614, a moderate increase in basal vir gene expression was observed as judged by virE::cat fusion expression assays relative to the wild-type control plasmid. Furthermore, higher efficiencies of tobacco leaf disk transformation were observed than with the widely used vir helper plasmid pAL4404. The nucleotide sequence studies showed that the vir region consists of 28,631 bp comprising 24 open reading frames which encode proteins involved in tumorigenicity. Two open reading frames not previously characterized, virH and ORF5, were uncovered within the virD/virE intervening spacer region. Together these studies more completely characterize the structure and function of the vir regulon.