Wild-type Promoter Region Research Articles

DNA watermarks in non-coding regulatory sequences

BackgroundDNA watermarks can be applied to identify the unauthorized use of genetically modified organisms. It has been shown that coding regions can be used to encrypt information into living organisms by using the DNA-Crypt algorithm. Yet, if the sequence of interest presents a non-coding DNA sequence, either the function of a resulting functional RNA molecule or a regulatory sequence, such as a promoter, could be affected. For our studies we used the small cytoplasmic RNA 1 in yeast and the lac promoter region of Escherichia coli.FindingsThe lac promoter was deactivated by the integrated watermark. In addition, the RNA molecules displayed altered configurations after introducing a watermark, but surprisingly were functionally intact, which has been verified by analyzing the growth characteristics of both wild type and watermarked scR1 transformed yeast cells. In a third approach we introduced a second overlapping watermark into the lac promoter, which did not affect the promoter activity.ConclusionEven though the watermarked RNA and one of the watermarked promoters did not show any significant differences compared to the wild type RNA and wild type promoter region, respectively, it cannot be generalized that other RNA molecules or regulatory sequences behave accordingly. Therefore, we do not recommend integrating watermark sequences into regulatory regions.

Human papillomavirus type 16 P670 promoter is negatively regulated by CCAAT displacement protein

HPV16 late gene transcription from P670 is suppressed in undifferentiated keratinocytes. To identify DNA sites involved in the negative regulation, we examined the effect of a series of substitutions in the P670 promoter region (nucleotide (nt) 106-855) on the transcription, using an expression plasmid having the promoter fragment placed to drive the firefly-luciferase gene. Twenty-base pair-long segments covering the entire promoter region were replaced with a sequence lacking any so far known factor-binding motifs to produce 38 mutants. These plasmids were introduced by transfecton into undifferentiated or partially differentiated human HaCaT and HeLa cells, and transient expression of the reporter was examined with the cell extracts. The reporter expression from the wild-type promoter region was lower, half to one-third, in the undifferentiated cells than in the partially differentiated cells, which expressed hSkn-1a, a keratinocyte specific transcription factor that activates P670, and CCAAT displacement protein (CDP), a transcriptional repressor involved in cell differentiation. Two mutants with substitutions including the putative CDP-binding sites, one from nt 562 to 567 and the other from nt 673 to 678, induced markedly enhanced reporter expression particularly in the partially differentiated cells. Electrophoretic mobility shift analysis demonstrated that bacterially produced GST-CDP bound to the two sites in a sequence-specific manner. The data strongly suggest that CDP acts as a major suppressor for P670 transcription by binding to the promoter region in the undifferentiated cells and even in the partially differentiated cells that express the activator hSkn-1a.

Site-directed mutagenesis of conserved inverted repeat sequences in the xylanase C promoter region from Streptomyces sp. EC3.

Streptomyces sp. EC3, a strain which was originally isolated from cattle manure compost, was shown to possess a strong xylanolytic activity. One of the genes responsible for this activity, xlnC, encodes a secreted xylanase. In the native strain, as in the heterologous host S. lividans, expression of xlnC was detectable in the presence of xylan but not in the presence of glucose. Induction by xylan was shown to take place at the transcriptional level. The transcriptional start site of xlnC was mapped and likely -35 (5'-TTGACA-3') and -10 (5'-GAGAAC-3') motifs were identified. In order to localise putative conserved regulatory sequences, the promoter regions of xylanase-encoding genes from various Streptomyces species were aligned. This alignment revealed the existence of three sets of quite well conserved palindromic AT rich sequences called boxes 1, 2 and 3. Box 3 (5'-CGAAA N TTTCG-3') is the farthest away from the promoter region (150-200 bp). A shorter version of this palindrome (5'-GAAA NN TTTC-3') or (5'-CGAAA-3') constitutes box 1, which is located just upstream of the putative -35 promoter sequence. Box 2, located 5-7 bp upstream of box 1, comprises a shorter palindrome than box 3, with inverted polarity [5'-(G/C)TTTC (N) GAAA(G/C)-3']. The putative regulatory role of the conserved inverted repeats in boxes 2 and 3 in the promoter region of the xlnC gene from Streptomyces sp. EC3, was assessed. These boxes were modified by site-directed mutagenesis, and the mutant promoter regions, as well as the wild-type promoter region, were separately fused to a beta-lactamase reporter gene. Analysis of the expression patterns of these fusions in cultures grown in the presence of glucose, xylan or both carbon sources demonstrated that these motifs were cis -acting negative regulatory elements, each playing a specific role in the regulation of xlnC expression. Box 3 was shown to be critical for the establishment of repression of xlnC expression by glucose, whereas box 2 was shown to play an important role in the induction of xlnC expression by xylan.

Effects of neighboring DNA homopolymers on the biochemical and physical properties of the Escherichia coli lactose promoter. III. High resolution thermal denaturation and circular dichroism studies.

High resolution thermal denaturation and circular dichroism studies were performed on a series of six recombinant DNA restriction fragments. The fragments varied in size from 132 to 193 bp and contained Escherichia coli wild type and UV5 lactose promoters both with and without homopolymer insertions of poly(dA).poly(dT) and poly(dG).poly(dC). A differential thermal destabilization of the wild type promoter region, as compared to the UV5 promoter, was observed when dA70.dT70 was inserted into the -60 region or both DNAs. This effect may depend, in part, on the differences in the base composition between adjoining cooperative units in the fragments. The relatively larger effect of the AT sequence on the wild type promoter region may be correlated with the increased levels of in vitro transcription activity described in the preceding paper (Klein, R. D., and Wells, R. D. (1982) J. Biol. Chem. 257, 12962-12969). Stretches of homopolymeric GC base pairs stabilized the wild type and UV5 promoter regions by over 2.5 degrees C. CD studies could not detect conformational differences between DNAs containing the wild type or UV5 promoter. The presence of homopolymers had a marked effect on the CD spectra of the fragments.